RU2795621C2 - Method for obtaining cells from dental pulp - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: invention relates to a method for obtaining cells derived from the dental pulp, enriched with pluripotent stem cells. To implement this method, first, the stage of hydrolysis of the dental pulp with collagenase and neutral metalloprotease is carried out to prepare a suspension of the dental pulp. This suspension is then cultured to proliferate the pluripotent stem cells contained in the suspension at 37°C in the presence of 5% CO₂ in DMEM supplemented with fetal calf serum at a concentration of 20% (v/v) and antibiotics, changing the medium every 2–4 days. Thereafter, the proliferated pluripotent stem cells are frozen in a state in which the pluripotent stem cells are suspended in the first cryopreservation liquid. The frozen pluripotent stem cells are then thawed. Thereafter, they are cultured in a state in which the pluripotent stem cells are attached to the surfaces of the carrier particles for the proliferation of the pluripotent stem cells on the surface of the carrier particles at 37°C in DMEM supplemented with 10% (v/v) fetal calf serum supplemented with glucose. Then, the step of bringing the carrier particles into contact with trypsin is carried out to separate the pluripotent stem cells attached to the surfaces of the carrier particles from the carrier particles, and the process is completed by the step of preparing a suspension of the separated pluripotent stem cells.

EFFECT: invention improves the efficiency of obtaining pluripotent stem cells from dental pulp and provides a novel preparation of pluripotent stem cells in a form that can maintain stability suitable for distribution and storage.

23 cl, 23 dwg, 25 tbl, 31 ex

Description

The field of technology to which the invention belongs

The present invention relates to pluripotent stem cells, which are obtained from the dental pulp and have the ability to differentiate into chondrocytes and osteocytes, as well as to a method for obtaining such pluripotent stem cells.

Prior Art

It is known that stem cells (pluripotent stem cells), which have the ability to differentiate into cells of many systems, can be obtained from various tissues. Mesenchymal stem cells isolated from bone marrow are one of these stem cells and have the ability to differentiate into various cells such as osteocytes, cardiomyocytes, chondrocytes, and adipocytes (Patent Literature 1-4). Attempts have been made to apply mesenchymal stem cells in regenerative medicine in various tissues using this ability to differentiate. For example, attempts have been reported to administer mesenchymal stem cells to patients with myocardial infarction to regenerate myocardium necrotized due to myocardial infarction, thereby improving the cardiac functions of patients (Non-Patent Literature 1).

It is known that mesenchymal stem cells can be obtained not only from bone marrow as described above, but also from various tissues such as adipose tissue (Patent Document 5), placental tissue and umbilical cord tissue (Patent Document 6).

Regarding the fact that pluripotent stem cells can also be obtained from dental tissue, it has been reported that pluripotent stem cells can be obtained from, for example, dental pulp (Patent Documents 7-11), dental follicle (Patent Literature 12), dental sac (Patent Document 11 and 13), dental papilla (Patent Document 14), and periodontal membrane (Patent Document 15). Pluripotent stem cells derived from dental tissues usually have the ability to differentiate into adipocytes (Non-Patent Literature 2-4).

Pluripotent stem cells derived from dental tissues usually have the ability to differentiate into adipocytes (Non-Patent Literature 2-4). The tissue obtained from the grinding of the extracted tooth is treated with type I collagenase and dispase (registered trademark) and cell clumps are removed by passing through a filter to obtain a cell suspension. The cells are then proliferated in a cell culture plate using DMEM containing 20% FBS. Cells that have adhered to the inner surface of the cell culture plate and proliferated are trypsinized and peeled off, and the detached cells are collected. The cells collected in this way are pluripotent stem cells of the dental pulp. Dispase is a neutral protease derived from Bacillus polymyxa.

The dental pulp is a loose fibrous connective tissue that fills the cavity of the dental pulp and is divided into crown pulp and root pulp, depending on its location. In addition, it has been reported that dental tissue-derived pluripotent stem cells generally have the ability to differentiate into adipocytes (Non-Patent Literature 2-4), while dental pulp-derived stem cells do not differentiate into adipocytes (Patent Literature 8). That is, there is a possibility that there are at least two types of stem cells derived from the dental pulp, which differ in the presence or absence of the ability to differentiate into adipocytes. That is, there is a possibility that there are at least two types of stem cells derived from the dental pulp, which differ in the presence or absence of the ability to differentiate into adipocytes. In addition, it has also been reported that dental pulp-derived pluripotent stem cells have properties different from those of bone marrow-derived mesenchymal stem cells regarding the induction of differentiation into osteoblasts (Patent Literature 11).

Pluripotent stem cells derived from dental pulp are expected to be clinically useful as they can be used as a therapeutic agent in neurological diseases (Patent Literature 17).

List of cited patent literature

Patent Literature 1: US Patent No. 5486359 Patent Literature 2: US Patent No. 5827740 Patent Literature 3: US Patent No. 6835377 Patent Literature 4: US Patent No. 6387369 Patent Literature 5: Japanese Unexamined Patent Publication No. 2004-129549.

Patent Literature 6: Japanese Patent Unexamined Publication No. 2004-210713.

Patent Literature 7: Japanese Unexamined Patent Publication No. 2010-252778.

Patent Literature 8: WO2002/007679.

Patent Literature 9: Japanese Patent Unexamined Publication No. 2008-507962.

Patent Literature 10: WO2009/072527.

Patent Literature 11: Japanese Patent Unexamined Publication No. 2004-201612.

Patent Literature 12: Japanese Patent Unexamined Publication No. 2009-527223.

Patent Literature 13: Japanese Patent Unexamined Publication No. 2005-516616.

Patent Literature 14: Japanese Unexamined Patent Publication No. 2006-238875.

Patent Literature 15: Japanese Patent Unexamined Publication No. 2008-295420.

Patent Literature 16. Japanese Patent Unexamined Publication No. 2010-268715.

Patent Literature 17: Japanese Patent Unexamined Publication No. 2011-219432.

Non-Patent Literature

Non-Patent Document 1: Katritsis DG. et. al., Catheter Cardiovasc Interv. 65(3): 321-9 (2005)

Non-Patent Literature 2: Gronthos S. et. al., J Dent Res. 81 (8): 531-5 (2002).

Non-Patent Literature 3: Tirino V. et. al., Stem Cell Rev. 7 (3): 608-15 (2011).

Non-Patent Literature 4: Vishwanath VR et. al., J Conserv Dent. 16 (5): 423-8 (2013).

Summary of the Invention Technical Problem

The purpose of the present invention is to provide a new preparation of pluripotent stem cells derived from dental pulp, which can be used to treat cerebral infarction and the like. and which can be administered to people. Another object of the present invention is to provide a method for efficiently obtaining pluripotent stem cells derived from dental pulp, so that pluripotent stem cells can be stably marketed in sufficient quantity. In addition, another object of the present invention is to obtain cells in a form in which stability can be maintained suitable for distribution or storage.

Solution

In studies for the above-described objects, the authors of the present invention conducted extensive studies. As a result, they found a way to efficiently obtain pluripotent stem cell-enriched dental pulp-derived cells from human extracted dental pulp tissue, and completed the present invention. That is, the present invention includes the following.

1. A method of obtaining cells derived from the dental pulp enriched with pluripotent stem cells, comprising the step of (a) hydrolyzing the dental pulp with a protease to prepare a suspension of the dental pulp; step (b) culturing the suspension to proliferate the pluripotent stem cells contained in the suspension; step (c) freezing the proliferated pluripotent stem cells in a state in which the pluripotent stem cells are suspended in the first cryopreservation liquid; step (d) thawing the frozen pluripotent stem cells; step (e) culturing the thawed pluripotent stem cells in a state in which the pluripotent stem cells are attached to the surfaces of the carrier particles to proliferate the pluripotent stem cells on the surface of the carrier particles; step (f) contacting the carrier particles with a protease to separate the pluripotent stem cells attached to the surfaces of the carrier particles from the carrier particles; and step (g) preparing a suspension of the separated pluripotent stem cells.

2. The production method according to 1 above, wherein the quality tests of the pluripotent stem cells are carried out in step (c) or (d).

3. The production method as described above 2, in which quality tests are performed on cells fractionated from cells before being suspended in the first cryopreservation liquid, subcultured cells that have been fractionated from cells before being suspended in the first cryopreservation liquid, pre-frozen cells that have been suspended and fractionated in the first cryopreservation liquid, cells immediately after thawing, which were fractionated and frozen, and/or cells obtained by fractionation, freezing, thawing and cultivation.

4. The production method as described above 3, wherein the quality tests are performed for one or more of the following quality tests a to j.

Quality test a, which checks that the proportion of the number of cells showing a substantially fusiform shape in all cells when observed with an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9%, or greater than or equal to 99.95%,

Quality test b, which checks that cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95%,

Quality test c that confirms that cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression patterns, or checks that cells are positive for at least one of CD73 and CD90 and negative for CD34 in expression patterns of cell surface antigen markers,

Quality test d, which checks that cells are negative for CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression patterns, or checks that cells are negative for at least one of CD40, CD80, CD86 and MHC class II antigen in expression patterns of cell surface antigen markers,

Quality test e, which checks that cells remain negative for CD40, CD80 and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression patterns when cells are stimulated with interferon-γ, or checks that at least any of the cells that are CD40 negative, CD80 negative, CD86 negative, or MHC class II antigen positive in cell surface antigen marker expression patterns when the cells are stimulated with interferon-γ,

quality test f, which checks the cell expression of prostaglandin E2 and/or vascular endothelial growth factor (VEGF), and the expression level of prostaglandin E2 is increased by stimulating TNF-α cells,

Quality test g, which confirms that the expression level of aggrecan increases when cells are cultured in a medium containing a substance that causes differentiation into chondrocytes,

The h quality test, which confirms that the amount of calcium accumulated in cells increases when cells are cultured in a medium containing a substance that induces osteocyte differentiation,

i quality test, which confirms that cells have the ability to divide at least 10, 14, or 15 times in a cell culture plate, and

Quality test j, which checks that the average cell doubling time in the cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours, or 36 hours during the quality testing period i.

5. The production method according to 1-4 above, further comprising: loading the suspension onto a filter membrane to collect cells that were skipped in step (g).

6. The production method as described above 5, wherein the filter membrane has a pore diameter of 20 µm to 80 µm.

7. The method of obtaining according to the above 1-6, in which the pulp is obtained from permanent or temporary teeth of a person.

8. The production method according to 1-7 above, wherein the protease used in step (a) comprises a serine protease, a metalloproteinase, or a mixture thereof.

9. The production method according to 1-7 above, wherein the protease used in step (a) comprises a metalloproteinase.

10. The preparation method according to 1-7 above, wherein the protease used in step (a) comprises a matrix metalloproteinase, a neutral metalloproteinase, or a mixture thereof.

11. The production method according to 1-7 above, wherein the protease used in step (a) includes collagenase and neutral metalloproteinase.

12. The production method as described above 11, wherein the collagenase comprises collagenase I, collagenase II, or a mixture thereof.

13. The production method according to 10 or 11 above, wherein the neutral metalloproteinase comprises thermolysin, dispase, or a mixture thereof.

14. The preparation method according to 1-13 above, wherein the first cryopreservation liquid used in step (c) comprises Ringer's bicarbonate solution, human serum albumin and DMSO.

15. The production method according to 1-14 above, wherein the carrier particles used in step (e) are substantially spherical in shape having a diameter of 80 to 300 µm in a swollen state.

16. The preparation method as described above 15, wherein the carrier particles are porous carrier particles having pores of 3 to 40 µm in diameter, which open to the surfaces of the carrier particles in a swollen state.

17. The preparation method according to 1-16 above, wherein the carrier particles contain gelatin.

18. The preparation method according to 1-17 above, wherein the protease used in step (f) comprises a serine protease, a metalloprotease, or a mixture thereof.

19. The production method according to 1-17 above, wherein the protease used in step (f) includes trypsin.

20. The production method according to any of 1 to 19 above, further comprising the step (h) of freezing the suspension obtained in step (g) in a state in which the suspension is in the second cryopreservation liquid.

21. The production method as described above 20, wherein the second cryopreservation fluid used in step (h) comprises Bicarbonate Ringer's solution, human serum albumin and DMSO.

22. The production method according to 1 to 19 above, wherein the quality tests of the pluripotent stem cells are carried out in step (g).

23. The production method according to 20 or 21 above, wherein the quality tests of the pluripotent stem cells are carried out in step (g) or (h).

24. The preparation method according to 22 or 23 above, in which quality tests are carried out on cells fractionated from cells before being suspended in the second cryopreservation liquid, subcultured cells that have been fractionated from cells before being suspended in the second cryopreservation liquid, pre-frozen cells , which were suspended in the second cryopreservation liquid and fractionated, cells immediately after thawing, which were fractionated and frozen, and/or cells obtained by fractionation, freezing, thawing and culturing.

25. The production method according to 22 or 24 above, wherein quality tests are performed for one or more of the following quality tests a' to j'.

Quality test a', which checks that the proportion of the number of cells showing a substantially fusiform shape in all cells when observed with an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9% or greater than or equal to 99.95%,

Quality test b', which checks that the cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95%,

Quality test c' that checks that the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression patterns, or checks that the cells are positive for at least one of CD73 and CD90 and CD34 negative in expression patterns of cell surface antigen markers,

Quality test d' that checks that cells are negative for CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression patterns, or checks that cells are negative for at least CD40, CD80, CD86 and MHC -class II antigen in expression patterns of cell surface antigen markers,

An e' quality test that checks that cells remain negative for CD40, CD80 and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression patterns when cells are stimulated with interferon-γ, or checks for at least any one of the cells that are CD40 negative, CD80 negative, CD86 negative, or MHC class II antigen positive in cell surface antigen marker expression patterns when the cells are stimulated with interferon-γ,

Quality test f, which checks that cells express prostaglandin E2 and/or vascular endothelial growth factor (VEGF) and the expression level of prostaglandin E2 is increased by stimulation of TNF-α cells,

The quality test g', which checks that the expression level of aggrecan is increased when cells are cultured in a medium containing a substance that induces differentiation into chondrocytes,

The h' quality test, which checks that the amount of calcium accumulated in cells is increased when the cells are cultured in a medium containing a substance that induces differentiation into osteocytes,

i' quality test, which checks the ability of cells to divide at least 3, 4 or 5 times in a cell culture plate, and

Quality test j', which checks that the average cell doubling time in the cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours, or 36 hours during the quality test period i'.

26. The production method as described above 22-25, wherein the percentage of CD107b positivity in the pluripotent stem cells used in the quality tests in step (g) or (h) is higher than the corresponding positivity rate of the pluripotent stem cells used in the tests quality in step (c) or (d).

27. The method of production according to any of the above 22-26, where the positive percentage of at least one of CD39, CD49a, CD61, CD107a, CD107b and CD143 in pluripotent stem cells used in quality tests at stage (g) or ( h) higher than the corresponding positive proportion of pluripotent stem cells used in the quality tests in step (c) or (d) and the positive proportion of CD146 in pluripotent stem cells used as tests in step (g) or (h) below, than the corresponding positivity rate for the pluripotent stem cells used in the quality tests in step (c) or (d).

28. The production method as described above 22-27, wherein the expression level of at least one of IL-6, HGF, IGFBP-4, IL-11, TIMP-3 and TIMP-2 in the pluripotent stem cells used in quality tests in step (g) or (h) is higher than the corresponding expression level in the pluripotent stem cells used in the quality tests in step (c) or (d).

29. Cells obtained by the method of obtaining according to any of the above 1-28.

30. Cells as described above 29 that are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45.

31. Cells as described above 30 that are negative for CD40 antigen, CD80, CD86 and MHC class II.

32. Cells as described above 31 that become positive for the MHC class II antigen when the cells are stimulated with interferon-γ.

33. Dental pulp-derived pluripotent stem cells having at least one of the characteristics shown in (1) to (4) below.

(1) cells positive for CD73, CD90, CD105 and CD166

and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen become positive for MHC class II antigen when cells are stimulated with interferon-γ and express prostaglandin E2 and/or vascular endothelial growth factor, and expression level prostaglandin E2 increases when cells are stimulated by TNF-α,

(2) the cells are positive for at least one of CD73, CD90, CD105, and CD166 and negative for CD34 and CD45,

3. the cells are positive for at least one of CD47, CD81 and CD147 and negative for at least one of CD19, CD34 and CD206, and

4. cells are positive for at least one of CD47, CD81 and CD147 and negative for at least one of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a and SSEA-1.

34. Cells as described above 33, which have the ability to differentiate into osteocytes and chondrocytes.

35. Cells as described above 33 or 34, wherein the average cell diameter in the state in which the cells are forced to float in the medium is 16 to 20 µm.

36. Cells according to any one of 33-35 above, which have the ability to divide at least 3 times in in vitro medium and in which the average doubling time is within 96 hours.

37. Dental pulp-derived pluripotent stem cells according to any of 33-35 above, which have the following characteristics: cells divide at least 10, 15, 16, or 17 times in an in vitro medium, and an average division time cells is within 48 hours, and the cells have the ability to divide at least 10, 14 or 15 times in the in vitro medium, and the average cell division time is within 96 hours, 84 hours, 72 hours, 48 hours or 36 hours.

38. Dental pulp-derived pluripotent stem cells according to any one of 33-35 above, which have the following characteristics: the cells have split at least 16, 21, 22, or 23 times in an in vitro medium, and the cells have the ability to divide at least 3, 4 or 5 times in the in vitro medium, and the average cell division time is within 96 hours, 84 hours, 72 hours, 48 hours or 36 hours.

39. Dental pulp-derived pluripotent stem cells, according to any of 33-38 above, which are negative for at least one of CD19, CD26, CD106, CD117, and CD271.

40. Dental pulp-derived pluripotent stem cells according to any of 33-39 above that are positive for at least one of CD140b and HLA-A, -B and -C.

41. Dental pulp-derived pluripotent stem cells, according to any of 33-40 above, which are negative for at least one of CD56 and CD146.

42. Pluripotent stem cells derived from dental pulp, according to any of 33-41 above, which are positive for at least one of CD49e and CD95.

43. Pluripotent stem cells derived from dental pulp, according to any of the above 33-42, which are positive for at least one of CD10, CD46, CD47, CD55, CD58 and CD59.

44. Pluripotent stem cells derived from dental pulp, according to any of the above 33-43, which express at least one of MMP-2, IGFBP-4, cystatin C, IL-6, IL-11, MCP -1, IL-8, HGF, VEGF, TIMP-1, TIMP-2 and TIMP-3.

45. Dental pulp-derived pluripotent stem cells, according to any of 33-44 above, which express at least one of GROα, VCAM-I, and IP-10.

46. Pluripotent stem cells derived from dental pulp, according to any of the above 33-45, which express IL-6 and in which its expression level is increased by stimulation with TNF-α and stimulation with interferon-γ.

47. Pluripotent stem cells derived from dental pulp, according to any of the above 33-46, which express IL-11 and in which its expression level is increased by stimulation with TNF-α and stimulation with interferon-γ.

48. Pluripotent stem cells derived from dental pulp, according to any of the above 33-47, which express IP-10 and in which its expression level is increased due to stimulation with TNF-α and stimulation with interferon-γ.

49. Dental pulp-derived pluripotent stem cells of any of the above 33-48, which express MCP-1 and are upregulated by TNF-α stimulation and interferon-γ stimulation.

50. Dental pulp-derived pluripotent stem cells, according to any of the above 33-49, in which GM-CSF expression is induced by TNF-α stimulation.

51. Pluripotent stem cells derived from dental pulp, according to any of the above 33-50, which express HGF and in which its expression level is reduced by TNF-α stimulation and increased by interferon-γ stimulation.

52. Dental pulp-derived pluripotent stem cells according to any of 33-51 above, which express IL-8 and in which its expression level is increased by TNF-α stimulation.

53. Dental pulp-derived pluripotent stem cells according to any one of 33-52 above, wherein the dental pulp is derived from human permanent or temporary teeth.

54. A composition comprising: dental pulp-derived pluripotent stem cells according to any of 33-53 above, suspended in Ringer's bicarbonate solution containing human serum albumin and dimethyl sulfoxide.

55. Composition containing: derived from dental pulp pluripotent stem cells according to any of the above 33-53, suspended in a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide .

56. The composition as described above 55, in which sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide are respectively contained in concentrations from 91 to 113 mm, from 2.52 to 3, 08 mM, 0.95 to 1.16 mM, 0.315 to 0.385 mM, 15.6 to 19.2 mM, 1.04 to 1.28 mM, 46 to 56 g/l and 9% up to 11% (v/v).

57. The composition as described above 54-56, additionally containing: acetyltryptophan or its salts; and caprylic acid or salts thereof.

58. A composition according to any one of 54-57 above, wherein the dental pulp-derived pluripotent stem cells are contained at a density of 5 x 10 ⁶ to 8 x 10 ⁷ cells/mL.

59. The composition as described above 54-58, which is encapsulated in a container in an amount of from 1 to 20 ml.

60. The composition as described above 59, in which the container is made of glass or plastic.

61. The composition as described above 54-60, which is in a frozen state.

62. A pharmaceutical composition comprising: a composition according to any one of 54-61 above.

63. The pharmaceutical composition as described above 62, which is a therapeutic agent for the treatment of a disease selected from the group consisting of autoimmune disease, inflammatory disease, rheumatoid arthritis, Crohn's disease, chronic inflammatory bowel disease, myocardial infarction, cerebral infarction (including chronic cerebral infarction and acute cerebral infarction), chronic inflammatory demyelinating polyneuritis, multiple sclerosis, systemic lupus erythematosus, cirrhosis of the liver (including decompensated cirrhosis of the liver), sepsis, osteoarthritis, psoriasis, and organ transplant rejection.

64. A pharmaceutical composition as described above 62 for use in the treatment of a disease selected from the group consisting of autoimmune disease, inflammatory disease, rheumatoid arthritis, Crohn's disease, chronic inflammatory bowel disease, myocardial infarction, cerebral infarction (including chronic cerebral infarction and acute cerebral infarction), chronic inflammatory demyelinating polyneuritis, multiple sclerosis, systemic lupus erythematosus, cirrhosis of the liver (including decompensated cirrhosis of the liver), sepsis, osteoarthritis, psoriasis, and organ transplant rejection.

Useful effects of the invention

According to the present invention, there is provided a new pluripotent stem cell preparation derived from dental pulp, which is useful in the treatment of cerebral infarction and the like. and can be administered to humans. In addition, according to the present invention, such dental pulp-derived cells enriched in pluripotent stem cells (dental pulp-derived cells enriched in pluripotent stem cells) that can be administered to humans can be efficiently produced, and therefore pharmaceuticals can be stably supplied. products containing these cells derived from dental pulp as active ingredients according to needs. In addition, since the cells derived from the dental pulp enriched in pluripotent stem cells can be obtained in a stable form, the possibility of deterioration during distribution or storage can also be avoided.

Brief description of the drawings

Fig. 1 is a graph showing the mean measurements of cell particle diameters of dental pulp-derived cells (intermediate cells and cells contained in dental pulp-derived cell preparations) that are respectively contained in intermediates and pulp-derived cell preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the average particle diameter (µm). The mean particle diameters of intermediate cells and cells contained in cell preparations derived from dental pulp are shown in order from left to right. Error bars indicate the standard deviation (n = 3).

Fig. 2 is a graph showing the results of testing the ability to differentiate into osteocytes in cells derived from dental pulp obtained in examples 7 and 16. The vertical axis indicates the concentration of calcium (µg/ml). For intermediate cells (Example 7) and cells in cell preparations derived from dental pulp (Example 16), black bars indicate the concentrations of calcium released from cells of the osteocyte differentiation-inducing groups, and white bars indicate the concentrations of calcium released from cells of the control groups. .

Fig. 3 is a graph showing the results of testing the ability to differentiate into chondrocytes in cells derived from dental pulp obtained in examples 7 and 16. The vertical axis indicates the Ct value of aggrecan (ACAN). With respect to intermediate cells and cells in dental pulp-derived cell preparations, black bars indicate aggrecan Ct values (ACAN) of cells of chondrocyte differentiation-inducing groups, and white bars indicate aggrecan Ct values (ACAN) of control group cells.

Fig. 4 is a graph showing the results of measuring the surface antigens of cells obtained from dental pulp, obtained in examples 7 and 16. The vertical axis indicates the proportion (%) of positive cells. For intermediate cells (black bars) and cells (white bars) in cell preparations derived from dental pulp, the proportions of positive cells for CD34, CD45, CD73, CD90, CD105, and CD166 are shown in order from left to right.

Fig. 5 is a graph showing the results of measuring the surface antigens of cells obtained from dental pulp (intermediate cells) contained in the intermediates obtained in Example 7. The vertical axis indicates the proportion (%) of positive cells. Proportion of positive cells for CD9, CD10, CD13, CD29, CD44, CD46, CD47, CD49b, CD49c, CD49f, CD55, CD58, CD59, CD63, CD73, CD81, CD90, CD98, CD140b, CD147, CD151, CD164, CD166 receptor epidermal growth factor (EGF-R) and human leukocyte antigen-A, -B and -C (HLA-A, -B and -C) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 9).

Fig. 6 is a graph showing the results of measuring the surface antigens of cells contained in the cell preparations obtained from the dental pulp obtained in Example 16. The vertical axis indicates the proportion (%) of positive cells. Proportion of positive cells for CD9, CD10, CD13, CD29, CD44, CD46, CD47, CD49b, CD49c, CD49e, CD49f, CD55, CD58, CD59, CD63, CD73, CD81, CD90, CD95, CD98, CD140b, CD147, CD151, CD164, CD166, EGF-R and HLA-A, -B and -C are shown in order from left to right. Values are means and error bars indicate standard errors (n = 7).

Fig. 7 is a graph showing the differences (percentage positive (%) in formulations - positive rate (%) in intermediates) between intermediates and formulations, respectively, obtained in Examples 7 and 16 for surface antigens having a positive rate difference of at least 10 % in cells derived from dental pulp contained in intermediate products and preparations. Results for CD39, CD49a, CD61, CD107a, CD107b, CD143 and CD146 are shown in order from left to right. Values are means and error bars indicate standard errors (n = 7).

Fig. 8 is a graph showing the results of testing the secretory capacity of VEGF cells derived from dental pulp in the intermediates and preparations respectively prepared in Examples 7 and 16. The vertical axis indicates the amount (pg/1×10 ⁵ cells) of secreted vascular endothelial growth factor ( VEGF).

Fig. 9 is a graph showing the results of the prostaglandin E2 (PGE2) secretory capacity test of dental pulp-derived cells in the intermediates and preparations respectively obtained in Examples 7 and 16. The vertical axis indicates the amount (pg/1×10 ⁵ cells) of secreted PGE2 .

Black bars indicate results in tumor necrosis factor (TNF-α) stimulated groups and white bars indicate results in tumor necrosis factor stimulated groups.

Fig. 10 is a graph showing the test results of kynurenine secretory capacity of dental pulp-derived cells obtained in Examples 7 and 16. The vertical axis shows the amount (ng/1 × 10 ⁵ cells) of kynurenine secreted by intermediate cells and cells in cell preparations obtained from the dental pulp.

Fig. 11A is a graph showing the results of the test for low immunogenicity of cells derived from dental pulp in the intermediates obtained in Example 7. The vertical axis indicates the proportion (%) of positive cells. Black bars indicate results in non-interferon-γ (IFN-γ) stimulated groups, and white bars indicate results in IFN-γ stimulated groups. Proportions of positive cells for major histocompatibility class I antigen (MHC), major histocompatibility class II antigen (MHC), CD40, CD80 and CD86 are shown in order from left to right.

Fig. 11B is a graph showing the results of the test for low immunogenicity of dental pulp-derived cells in the preparations prepared in Example 16. The vertical axis indicates the proportion (%) of positive cells. Black bars indicate results in non-IFN-γ challenged groups and white bars indicate results in IFN-gamma challenged groups. Proportions of positive cells for MHC class I antigen, MHC class II antigen, CD40, CD80 and CD86 in order from left to right.

Fig. 12 is a graph showing the results of measuring the concentrations of various factors such as cytokine in culture supernatants when cells derived from dental pulp contained in the intermediates and preparations respectively obtained in Examples 7 and 16 are not stimulated. The vertical axis shows the concentration (pg/1 x 10 ⁵ cells) of various factors. Concentrations of matrix metalloprotease-2 (MMP-2), insulin-like growth factor-binding protein-4 (IGFBP-4), and cystatin C for intermediate cells (black bars) and cells (white bars) in cell preparations derived from dental pulp are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 13 is a graph showing the results of measuring concentrations of various factors such as cytokine in culture supernatants when cells derived from dental pulp contained in the intermediates and preparations respectively obtained in Examples 7 and 16 are not stimulated. The vertical axis shows the concentration (pg/1 x 10 ⁵ cells) of various factors. Concentrations of interleukin-6 (IL-6), interleukin-11 (IL-11), monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), human growth regulated protein alpha (GROα), hepatocyte factor growth factor (HGF), vascular endothelial growth factor (VEGF), vascular endothelial adhesion molecule type 1 (VCAM-1), tissue inhibitor-3 metalloproteinases (TIMP-3), tissue inhibitor-2 metalloproteinases (TIMP-2), and tissue inhibitor -1 metalloproteinases (TIMP-1) for intermediate cells (black bars) and cells (white bars) in dental pulp-derived cell preparations are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 14 is an image showing the results of measuring concentrations of various factors such as cytokine in culture supernatants when cells derived from dental pulp contained in the intermediates and preparations respectively obtained in Examples 7 and 16 are not stimulated. The vertical axis shows the concentration (pg/1 x 10 ⁵ cells) of various factors. Concentrations of interleukin-23 (IL-23), interleukin-21 (IL-21), interferon-α (IFN-α), TNF-α, interleukin-18 (IL-18), interleukin-33 (IL-33), interleukin-27 (IL-27), thymus-activated chemokine (TARC), epithelial neutrophil-activating protein-78 (ENA-78), macrophage inflammatory protein-3a (MIP-3α), macrophage inflammatory protein-1p (MIP -1β), eotaxin, interferon-γ-inducible protein-10 (IP-10), macrophage inflammatory protein-1a (MIP-1a), interferon-gamma-induced monokine (MIG), interferon-induced T cell chemoattractant alpha (I-TAC), stem cell factor (SCF), granulocyte-monocyte colony stimulating factor (GM-CSF), and intercellular adhesion molecule-1 (ICAM-1) for intermediates (black bars) and drugs (white bars) are displayed in order from the left right. Values are means and error bars indicate standard errors (n = 3).

Fig. 15 is a graph showing the ratios (preparation concentration/intermediate concentration) of factors whose concentrations in the preparation are higher than those in the intermediate among various factors such as cytokine in culture supernatants when cells derived from dental pulp contained in intermediates and drugs, respectively obtained in examples 7 and 16, are not stimulated.

Concentration ratios of IL-6, IL-23, IL-11, MCP-1, ENA-78, HGF, VEGF, MMP-2, IGFBP-4, Cystatin C, TIMP-3, TIMP-2 and TIMP-1 are displayed in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 16 is a graph showing the concentrations of IL-6 in culture supernatants in the groups without stimulation, TNF-α stimulation, and IFN-γ stimulation for pulp-derived cells contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Concentrations in unstimulated groups, TNF-α stimulated groups, and IFN-γ stimulated groups for intermediates (black bars) and drugs (white bars) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 17 is a graph showing the concentrations of IL-11 in culture supernatants in the groups without stimulation, TNF-α stimulation and IFN-γ stimulation for pulp-derived cells contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Results for unstimulated groups, TNF-α stimulated groups, and IFN-γ stimulated groups for intermediates (black columns) and drugs (white columns) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 18 is a graph showing the concentrations of IP-10 in culture supernatants in the groups without stimulation, TNF-α stimulation and IFN-γ stimulation for cells derived from dental pulp contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Results for unstimulated groups, TNF-α stimulated groups, and IFN-γ stimulated groups for intermediates (black columns) and drugs (white columns) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 19 is a graph showing the concentrations of MCP-1 in culture supernatants in the groups without stimulation, TNF-α stimulation, and IFN-γ stimulation for pulp-derived cells contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Results for unstimulated groups, TNF-α stimulated groups, and IFN-γ stimulated groups for intermediates (black columns) and drugs (white columns) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 20 is a graph showing the concentrations of GM-CSF in culture supernatants in the groups without stimulation, TNF-α stimulation and IFN-γ stimulation for pulp-derived cells contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Results in unstimulated groups, TNF-α-stimulated groups, and IFN-γ-stimulated groups for intermediates (black bars) and drugs (white bars) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 21 is a graph showing the concentrations of HGF in culture supernatants in the groups without stimulation, TNF-α stimulation and IFN-γ stimulation for cells derived from dental pulp contained in the intermediates and preparations, respectively, obtained in examples 7. and 16. Vertical the axis indicates the concentration (pg/1 x 10 ⁵ cells). Results in unstimulated groups, TNF-α-stimulated groups, and IFN-γ-stimulated groups for intermediates (black bars) and drugs (white bars) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Fig. 22 is a graph showing the concentrations of IL-8 in culture supernatants in the groups without stimulation, TNF-α stimulation, and IFN-γ stimulation for pulp-derived cells contained in the intermediates and preparations, respectively, obtained in Examples 7 and 16. The vertical axis indicates the concentration (pg/1 x 10 ⁵ cells). Results in unstimulated groups, TNF-α-stimulated groups, and IFN-γ-stimulated groups for intermediates (black bars) and drugs (white bars) are shown in order from left to right. Values are means and error bars indicate standard errors (n = 3).

Description of embodiments

A pluripotent stem cell is a cell that has the ability to self-replicate and differentiate into two or more cells. Pluripotent stem cells derived from human pulp, which can be obtained using the present invention, preferably have the ability to differentiate into chondrocytes and osteocytes, but the present invention is not specifically limited to this.

The fact that the pluripotent stem cell that can be obtained using the present invention has the ability to differentiate into chondrocytes can be confirmed, for example, by quantifying the expression level of the aggrecan gene when the cell is cultured in a medium containing a substance known to induces cell differentiation into a chondrocyte. Aggrecan is the main molecule constituting the extracellular matrix of cartilage, and its expression level increases when cell differentiation into chondrocytes is induced. In this measurement method, TGF-b3 can be used as a substance (inducer of chondrocyte differentiation), which, for example, induces differentiation into chondrocyte cells. The measurement method described in Example 20 is a suitable example of a method for measuring the ability to differentiate into chondrocytes.

The fact that the pluripotent stem cell obtainable by the present invention has the ability to differentiate into osteocytes can be confirmed, for example, by quantifying the amount of calcium accumulated in the cell when the cell is cultured in a medium containing a substance which, as known to induce cell differentiation into osteocyte. When differentiation into osteocytes is induced, calcium accumulates in the cell. In this measurement method, dexamethasone, P-glycerophosphate, glycosaminoglycan, ascorbic acid, osteogenic factor 2 (BMP-2) and their salts can be used alone or in combination of two or more of them as a substance (inducer of osteocyte differentiation) that causes differentiation such as osteocytes.

For example, a combination of dexamethasone, ascorbate and P-glycerophosphate can be suitably used as an osteocyte differentiation inducing agent. The measurement method described in Example 19 is a suitable example of a method for measuring the ability to differentiate into osteocytes.

"Dental pulp-derived cells enriched in pluripotent stem cells" or "dental pulp-derived cells enriched in pluripotent stem cells" in the present invention are derived from dental pulp and cell aggregates in which the proportion (quantitative proportion) of pluripotent stem cells is higher, than in cells directly harvested from dental pulp, and includes pluripotent stem cells finally selectively isolated from dental pulp by culture or the like. Cells directly harvested from dental pulp are cells in which pluripotent stem cells are mixed with other cells. When these cells are cultured, the proportion of pluripotent stem cells at the end of culture is higher compared to that at the beginning of culture, because pluripotent stem cells have a higher proliferating ability than other cells. Since the proportion of pluripotent stem cells is further increased by repeated culturing, cells containing almost only pluripotent stem cells can be obtained. That is, “dental pulp-derived cells enriched in pluripotent stem cells” includes cells in which the proportion of pluripotent stem cells in all cells is higher than in cells immediately after harvesting from the dental pulp, due to the proliferation of pluripotent stem cells during the culture process. and so on. Human dental pulp-derived cells are specifically referred to as "human dental pulp-derived cells enriched in pluripotent stem cells".

In the present invention, pluripotent stem cells directly isolated from dental pulp and pluripotent stem cells contained in cells derived from dental pulp enriched in pluripotent stem cells by culture are collectively referred to as "dental pulp-derived pluripotent stem cells", and they are sometimes simply referred to as "dental pulp-derived stem cells" or "dental pulp stem cells". Human dental pulp-derived pluripotent stem cells are specifically referred to as "human dental pulp-derived pluripotent stem cells", or sometimes simply referred to as "human dental pulp-derived stem cells" or "human dental pulp stem cells". "Cells contained in intermediates (intermediate cells)" and "cells contained in cell preparations derived from dental pulp", which are one embodiment presented in the present description, essentially contain only pluripotent stem cells, and both are pluripotent stem cells. cells obtained from the dental pulp.

In the present invention, when a test population of cells is positive for certain antigens in expression patterns of surface antigen markers, this means that preferably at least 30%, more preferably at least 40%, even more preferably, at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and particularly preferably, at least 95% of the observed cells are positive for antigens when the cell population is observed as a whole. Expression patterns of surface antigens can be examined, for example, using the method described in Example 21 or 22, but the present invention is not limited to this.

In the present invention, in the case where the test population of cells is negative for certain antigens in the expression patterns of surface antigen markers, this means that preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, even more preferably less than 2% and particularly preferably, less than 1% of the observed cells are positive for antigens when the cell population is observed as a whole. Expression patterns of surface antigens can be examined, for example, using the method described in Example 21 or 22, but the present invention is not limited to this.

When the pluripotent stem cell-enriched dental pulp cells of the present invention are cultured in a medium containing a substance known to induce differentiation into chondrocytes, an increase in the expression level of aggrecan as a whole is observed. In addition, when the pluripotent stem cell-enriched cells of the present invention, derived from dental pulp, are cultured in a medium containing a substance known to induce differentiation into osteocytes, an increase in the amount of calcium accumulated in the cells is generally observed. That is, the pluripotent stem cell-enriched dental pulp-derived cells of the present invention have the ability to differentiate into chondrocytes and osteocytes when observed as a whole.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells of the present invention are observed as a whole (population), the cells are positive for at least one of CD73, CD90, CD105, and CD166 in marker expression patterns. surface antigens. Similarly, pluripotent stem cell-enriched dental pulp-derived cells of the present invention are negative for at least one of CD34 and CD45. For example, when pulp-derived cells enriched in pluripotent stem cells of the present invention are observed in general, the cells are, for example, positive for CD73 and CD90 and negative for CD34 in surface antigen marker expression patterns. In addition, when pulp-derived cells enriched in pluripotent stem cells of the present invention are observed in general, these cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in surface antigen marker expression patterns. These expression patterns are common to mesenchymal stem cells.

In addition, in one embodiment, when the cells of the present invention, derived from dental pulp enriched in pluripotent stem cells, are observed in general, the cells are negative for at least one of CD40, CD80, CD86 and MHC class II antigen in expression patterns of surface antigen markers. For example, when pulp-derived cells enriched with the pluripotent stem cells of the present invention are observed in general, the cells are negative for CD40, CD80, CD86 and MHC class II antigen in surface antigen marker expression patterns. It is known that when cells positive for these surface antigen markers are transplanted into allogeneic individuals, the cells tend to be recognized as an antigen to be eliminated from the living body. In the case where the cells are negative for at least one of these surface antigen markers, this means that the pulp-derived pluripotent stem cells of the present invention have the property of low immunogenicity to that extent and are unlikely to be eliminated from a living body when transplantation to allogeneic individuals. In addition, cells can remain negative for CD40, CD80 and CD86 and be positive for the MHC class II antigen in surface antigen marker expression patterns when the cells are stimulated with IFN-γ. For example, cells remain negative for CD40, CD80, and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells of the present invention are observed in general, these cells are, for example, positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen in expression patterns of surface antigen markers. During this time, for example, cells remain negative for CD40, CD80, and CD86 and may become positive for the MHC class II antigen when the cells are stimulated with IFN-γ.

In one embodiment, with respect to characteristic (a-1), when the dental pulp-derived cells enriched in pluripotent stem cells (dental pulp-derived pluripotent stem cells) of the present invention are observed in general, the cells are positive for at least one at a time, preferably all of CD29, CD46, CD47, CD59, CD73, CD81, CD90, CD147 and HLA-A, -B and -C in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (a-2), when the cells obtained from the dental pulp, enriched with pluripotent stem cells of the present invention are observed in general, these cells are positive in at least one, preferably in all of CD9, CD44, CD49b, CD49c, CD55, CD98 and EGF-R in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 65%, more preferably at least 75%, and even more preferably at least 80% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (a-3), when the cells obtained from the dental pulp enriched with pluripotent stem cells of the present invention are observed as a whole, the cells are positive in at least one, preferably in all of the numbers of CD49f, CD140b, and CD166 in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 70%, and even more preferably at least 75% of the observed cells are positive for these antigens .

In addition, regarding the characteristic (a-4), when the cells obtained from the dental pulp enriched in pluripotent stem cells of the present invention are observed in general, these cells are positive for at least one, preferably for all of CD10, CD13, CD58, CD63, CD105, CD151 and CD164 in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 65%, and even more preferably at least 70% of the observed cells are positive for these antigens.

In one embodiment of the invention, the pluripotent stem cell-enriched pulp-derived cells of the present invention preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in (a-1) above, (a -2), (a-3) and (a-4). For example, cells having the characteristics shown in (a-1) and (a-2) described above are a suitable embodiment of the present invention.

In one embodiment, regarding characteristic (a-5), when the cells derived from dental pulp enriched with pluripotent stem cells of the present invention are observed in general, these cells are negative for at least one, preferably for all of CD120b , CD132, CD158a, CD161, CD184, CD195, CD206, CD210, CD212, CD226, CD244, CD267, CD278, CD279, CD282, CD294, stage specific embryonic antigen-1 (SSEA-1), TRA-1-60, stage-specific embryonic antigen-3 (SSEA-3), skin lymphocyte-associated antigen (CLA), and β7 integrin in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably no more than 10%, more preferably no more than 5%, and even more preferably no more than 2%, and even more preferably no more than 1% of the observed cells are positive for these antigens.

In one embodiment, with respect to characteristic (a-6), when overall pulp-derived cells enriched in pluripotent stem cells of the present invention are observed, the cells are negative for at least one, preferably all, of CD8b, CD11b CD15s CD16 CD19 CD24 CD31 CD32 CD62E CD62P CD66f CD86 CD88 CD94 CD100 CD103 CD114 CD117 CD118 CD121b CD122 CD123 CD124 CD126 CD127 CD128b CD135, CD137, CD137, CD150, CD163, CD172b, CD177, CD178, CD180, CD197, CD220, CD229, CD231, CD255, CD268, CD305, CD314, CD321, CDw327, CDw328, CD329, CD335, CD336, leuco triene receptor B4 (BLTR-1), CLIP, CMRF-44, CMRF, CMRF-56, N-formylmethionyl leucylphenylalanine receptor (fMLP-R), invariant NKT, and γδ T-cell receptor (γδ TCR) in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably no more than 10%, more preferably no more than 5%, and even more preferably no more than 2% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (a-7), when the cells derived from dental pulp enriched with pluripotent stem cells of the present invention are observed in general, these cells are negative for at least one, preferably for all of CD1a , CD1b, CD1d, CD2, CD3, CD5, CD6, CD7, CD8a, CD11c, CD15, CD18, CD21, CD22, CD23, CD25, CD26, CD27, CD28, CD33, CD34, CD35, CD37, CD38, CD40, CD41a , CD41b, CD42b, CD45, CD45RB, CD45RO, CD48, CD50, CD53, CD62L, CD64, CD66 (a, c, d, e), CD69, CD70, CD72, CD80, CD84, CD85, CD87, CD89, CDw93 , CD97, CD106, CD134, CD138, CD144, CD154, CD158b, CD162, CD183, CD205, CD235a, CD271, CD309, CD326, CD337, αβ T cell receptor (αβ TCR) and MHC class II antigen in expression patterns of surface antigen markers .

Herein, when individual cells are observed included in all cells, preferably only at most 10% and more preferably only at least 5% of the observed cells are positive for these antigens.

In one embodiment of the invention, the pulp derived cells enriched in pluripotent stem cells of the present invention preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in (a-1) above, (a -2), (a-3) and (a-4), (a-5), (a-6) and (a-7). For example, cells having the characteristics shown in (a-1) and (a-5) described above are a suitable embodiment of the present invention.

In one embodiment of the present invention, when pulp-derived cells of the present invention enriched in pluripotent stem cells are observed in general, these cells express, for example, prostaglandin E2 (PGE2) and/or endothelial growth factor (VEGF), and the expression level of prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells. Since VEGF is an angiogenic substance, cells derived from dental pulp enriched with pluripotent stem cells can promote the formation of blood vessels in the body using VEGF by administering these cells to humans. Therefore, the cells can be used as a therapeutic agent for diseases in which an angiogenic effect is desired. In addition, since PGE2 has a strong anti-inflammatory effect, pluripotent stem cell-enriched dental pulp-derived cells act on the inflammatory site in the body through PGE2, and can suppress tissue destruction accompanied by inflammation by administering the cells to humans. Therefore, the cells can be used as an inhibitor of tissue destruction accompanied by inflammation. However, the effects of dental pulp cells enriched with pluripotent stem cells are not limited to the use of VEGF or PGE2.

In one embodiment of the present invention, when pluripotent stem cell-enriched dental pulp-derived cells are cultured in the presence of IFN-γ, the amount of kynurenine secreted increases. Kynurenine can inhibit T cell proliferation and differentiate monocytes into anti-inflammatory M2 macrophages. Accordingly, dental pulp-derived cells enriched in pluripotent stem cells can exert anti-inflammatory effects using kynurenine when administered to humans.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed in general, these cells are positive for at least one or all of CD47, CD81 and CD147 and negative for at least for one or all of CD19, CD34 and CD206 in surface antigen marker expression patterns. Further, in one embodiment of the present invention, when pluripotent stem cell-enriched dental pulp-derived cells are observed in general, these cells are positive for at least one or all of CD47, CD81, and CD147 and negative for at least one or all of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a, and SSEA-1 in surface antigen marker expression patterns.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed as a whole, these cells are negative for at least one, e.g., all of CD19, CD26, CD106, CD117, and CD271 in expression patterns of surface antigen markers.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed in general, these cells are positive for at least one, e.g., all of CD140b and HLA-A, -B and -C in expression patterns of surface antigen markers.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed in general, these cells are positive for at least one, for example, all of CD10, CD46, CD47, CD55, CD58, and CD59 in expression patterns of surface antigen markers.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed in general, the cells are, for example, positive for at least one of CD73, CD90, CD105, and CD166. and negative for at least one of CD34 and CD45 in surface antigen marker expression patterns.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells are observed in general, the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and the MHC- class II in expression patterns of surface antigen markers. Thus, for example, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC-class II antigen when the cells are stimulated with IFN-γ. Cells express prostaglandin E2 and/or vascular endothelial growth factor (VEGF), and the amount of secreted prostaglandin E2 is increased by stimulation of TNF-α cells. In addition, the amount of secreted kynurenine is increased by stimulation of IFN-γ cells.

When cells derived from dental pulp enriched with the pluripotent stem cells of the present invention are cultured, the cells secrete various humoral factors.

(a-8) In one embodiment, when pluripotent stem cell-enriched dental pulp-derived cells are cultured, the cells express at least one, preferably all, of MMP-2, IGFBP-4, and cystatin C.

(a-9) In one embodiment, when pluripotent stem cell-enriched dental pulp-derived cells are cultured, the cells express at least one, preferably all, of IL-6, IL-11, MCP-1, IL-8, GROα, HGF, VEGF, VCAM-I, TIMP-3, TIMP-2 and TIMP-1.

In addition, with respect to characteristic (a-10), when dental pulp-derived cells enriched in pluripotent stem cells are cultured, the cells express at least one, preferably all of IL-23, TNF-α, IL-18, IL-33, IL-27, TARC, ENA-78, MIP-3α, MIP-1β, IP-10, SCF and ICAM-1.

In addition, with regard to characteristic (a-11), in one embodiment, when cells derived from dental pulp enriched with pluripotent stem cells are cultured, these cells do not express or almost do not express at least one, preferably all of IL- 21, eotaxin, MIP-1a, MIG, I-TAC and GM-CSF.

In addition, regarding characteristic (a-12), in one embodiment, in the case where cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-6 is higher than in the case of cell culture in the absence of such.

In addition, regarding characteristic (a-13), in one embodiment, in the case where cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-11 is higher than in the case of cell culture in the absence of such.

In addition, regarding characteristic (a-14), in one embodiment, in the case where cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α or IFN-γ, the expression level of IP-10 is higher than in the case of cell culture in the absence of such.

In addition, regarding characteristic (a-15), in one embodiment, in the case where cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α or IFN-γ, the expression level of MCP-1 is higher than in the case of cell culture in the absence of such.

In addition, with respect to feature (a-16), in one embodiment, GM-CSF expression is induced when pluripotent stem cell-enriched dental pulp-derived cells are cultured in the presence of TNF-α, but GM-CSF expression is not induced when cells cultivated in the presence of IFN-γ.

In addition, with respect to characteristic (a-17), in one embodiment, the expression level of HGF is reduced when cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α compared to the case where the cells are cultured in its absence, while the expression level of HGF is increased when cells are cultured in the presence of IFN-γ compared to when cells are cultured in its absence.

In addition, with respect to characteristic (a-18), in one embodiment, when cells derived from dental pulp enriched in pluripotent stem cells are cultured in the presence of TNF-α, the expression level of IL-8 is greater than when cells are cultured in its absence.

In one embodiment of the invention, the pluripotent stem cell-enriched dental pulp-derived cells of the present invention preferably have two or more, more preferably three or more, and even more preferably four or more, and even more preferably all characteristics are shown in those described above ( a-8) - (a-18). For example, cells having the characteristics shown in (a-8) and (a-14) described above, or cells having the characteristics shown in (a-8), (a-13) and (a-14) described above. represent a suitable embodiment of the present invention.

The dental pulp-derived cells enriched in pluripotent stem cells of the present invention can be generally referred to as dental pulp-derived pluripotent stem cells. In many cases, dental pulp-derived pluripotent stem cells in squamous culture are observed under an optical microscope as attached spindle-shaped cells in a cell culture plate. However, they are not always observed as spindle-shaped attached cells depending on culture conditions.

"Dental pulp" in the present invention refers to loose fibrous connective tissue that fills the dental pulp cavity and is composed of: connective tissue containing blood vessels, nerves and lymphatic vessels; and an odontoblast layer capable of depositing and restoring within the dentin at the lateral margin. In addition, dental pulp can be divided into crown pulp and root pulp, depending on its location. The dental pulp in the present invention refers to a pulp including at least one of crown pulp and root pulp. In addition, animal species from which the dental pulp used in the present invention is obtained are not particularly limited, but are preferably mammals, more preferably primates, and even more preferably humans.

The pulp teeth may be permanent teeth or deciduous teeth, or may be any of incisors, canines, premolars and molars. The teeth are preferably healthy teeth not affected by caries or the like. For example, in the present invention, healthy teeth that have been removed by a medical procedure can be suitably used. In particular, third molars can be used particularly preferably because the amount of dental pulp that can be obtained from each tooth is large, and the teeth are easily accessible as extracted healthy teeth for reasons such as tooth correction or the like. In this document, the age of people who obtain teeth is not particularly limited. However, teeth obtained from people between the ages of 10 and 50 are preferred, and teeth obtained from people between the ages of 15 and 45 are more preferred. In addition, the sex of the people from which the teeth are obtained is not particularly limited.

To obtain dental pulp, it is preferable to use teeth within 72 hours of tooth extraction, more preferably use teeth within 24 hours of tooth extraction, and even more preferably use teeth within 12 hours of tooth extraction. The extracted teeth are washed with Ringer's bicarbonate solution or the like, and then sterilized with a sterilizing agent. The sterilizer currently used is not particularly limited, but one that exhibits an antibacterial effect against both Gram-positive bacteria and Gram-negative bacteria is preferred. For example, a 1% chlorhexidine gluconate solution is used.

The pulp of a tooth can be obtained from an extracted tooth by exposing the pulp by mechanical grinding of the extracted tooth. The dental pulp extracted from the extracted tooth is preferably crushed with a tool such as scissors and then processed with a protease.

By hydrolysis by a protease, the individual cells that make up the dental pulp are released from the pulp obtained from the extracted tooth. The currently used protease preferably comprises a serine protease, a metalloprotease, or a mixture thereof. However, the present invention is not limited to the above, and any protease that can release individual cells constituting the dental pulp, that is, that can degrade cell adhesion protein molecules, can be used.

Examples of metalloproteinases suitable for current use include matrix metalloproteinase, neutral metalloproteinase, or a mixture thereof. Collagenase, gelatinase, or a mixture thereof may suitably be used as the matrix metalloproteinase. In addition, collagenase I, collagenase II, or a mixture thereof is preferred as the collagenase. Thermolysin, dispase, or a mixture thereof can be suitably used as the neutral metalloproteinase.

Examples of a preferred combination of proteases include a mixture of collagenase I, collagenase II and thermolysin. When this mixture is used as a protease, the activity of the protease in the reaction solution is preferably adjusted to 0.17-1.33 U/ml. Liberase (registered trademark) (Roche) containing collagenase I, collagenase II and thermolysin can be suitably used as a protease.

In the case where a mixed solution of type II collagenase and Dispase is used, the respective concentrations of type II collagenase and Dispase are preferably 1 to 2 mg/mL and 3000 to 7000 units/mL, and more preferably about 1.5 mg/mL and about 5000 units/ml.

A suitable reaction temperature in the hydrolysis of dental pulp obtained from an extracted tooth using a protease is 35° C. to 37.5° C., and a suitable reaction time is 30 minutes to 3 hours. For example, the dental pulp is treated with a protease for 2 hours at 37°C. However, the reaction temperature and reaction time must be appropriately adjusted depending on, for example, the type and concentration of the protease.

Since the individual cells that make up the protease hydrolysed dental pulp are released from the dental pulp, the dental pulp can be disassembled by mechanical means such as pipetting. At this time, the protease is preferably inactivated before disassembling the dental pulp by mechanical means. Inactivation can be carried out by adding components that inactivate proteases to the reaction solution, and as such components, a chelating agent such as EDTA, a cell culture medium containing FBS, and the like can be used. By disassembling the dental pulp, a suspension can be obtained in which the cells and the like constituting the dental pulp are present in solution in a floating state. This suspension contains pluripotent stem cells released from the dental pulp. In order to remove the protease along with the supernatant, it is preferable to centrifuge this suspension once to precipitate insoluble substances such as cells. Precipitated cells, etc. resuspended after removal of the supernatant. Cell culture medium containing FBS can be used, for example, as a resuspension solution. The dental pulp suspension thus obtained includes not only cells released from the dental pulp, but also pieces of dental pulp tissue.

A suspension of dental pulp obtained by suspending disassembled dental pulp in a cell culture medium is added to a cell culture plate and cultured therein. The cell culture medium used for this is preferably Dulbecco's modified Eagle's medium to which fetal bovine serum is added. The concentration (v/v%) of fetal bovine serum added to the medium is preferably 8% to 25% and more preferably 10% to 25%, and is, for example, 20%. An example of a detailed composition of Dulbecco's modified Eagle's medium (prior to the addition of fetal calf serum) is shown in Table 1. Accordingly, 3 to 5 mM, eg 4 mM, L-alanyl-L-glutamine can be added to the medium. In addition, if desired, an antibiotic such as streptomycin can be added to the medium. When streptomycin is added to the medium, its concentration in the medium is set from 10 mg/l to 250 mg/l, for example 100 mg/l. In addition, each component can be replaced by its equivalent, such as its salt.

In the case of culturing a suspension of dental pulp obtained from one extracted tooth, such as the third molar of an adult, in a cell culture plate, the culture area in the plate is preferably 0.3 to 4 cm2 and more preferably 0.6 to 2 cm2 and is, for example, 1 cm2. The amount of medium added to the cell culture plate is preferably 250 µl to 1 ml per 1 cm2 culture area, for example 500 µl. For example, a suspension of dental pulp obtained from the third molar of an adult and suspended in a medium of 500 µl is added to a cell culture plate having a culture area of 1 cm2 for cell culture.

Table 1 Composition of Dulbecco's Modified Eagle's Medium

Component mM (range) mm (suitable example) Aminoacetic acid 0.2–0.6 0.4 L-arginine hydrochloride 0.3–0.5 0.398 L-cystine dihydrochloride 0.15–0.25 0.201 L-glutamine 3–5 4 L-histidine hydrochloride 0.15–0.25 0.2 monohydrate L-isoleucine 0.65–0.95 0.802 L-leucine 0.65–0.95 0.802 L-lysine hydrochloride 0.65–0.95 0.798 L-methionine 0.15–0.25 0.201 L-phenylalanine 0.2–0.6 0.4 L-serine 0.2–0.6 0.4 L-threonine 0.65–0.95 0.798 L-tryptophan 0.06–0.1 0.0784 L-tyrosine disodium 0.2–0.6 0.398 dihydrate L-valine 0.65–0.95 0.803 Choline chloride 0.02–0.04 0.0286 D-calcium pantothenate 0.007–0.009 0.00839 Folic acid 0.008–0.01 0.00907 Nicotinamide 0.03–0.05 0.0328 Pyridoxine hydrochloride 0.015–0.025 0.0196 Riboflavin 0.0008–0.0012 0.00106 Thiamine hydrochloride 0.01–0.014 0.0119 i-inositol 0.03–0.05 0.04 Calcium chloride 1.5–2.1 1.8 (anhydride) Iron(III) nitrate nonahydrate 0.0002–0.0004 0.000248 Magnesium sulfate 0.65–0.95 0.814 potassium chloride 5–6 5.33 sodium bicarbonate 40–48 44.05 Sodium chloride 100–120 110.34 Monohydrate 0.65–0.95 0.906 sodium dihydrogen phosphate D-glucose 5-7 5.56 Phenol red 0.03–0.05 0.0399 sodium pyruvate 0.8–1.2 1

The temperature at which the dental pulp suspension is cultured in the cell culture plate is preferably 35°C to 37.5°C, such as 37°C.

Cultivation of the dental pulp suspension is carried out, for example, until the cells begin to proliferate to form a visible size colony in the plate. The colony at this time forms a substantially circular shape when viewed from above and is about 1 to 3 mm in diameter. However, the present invention is not particularly limited to this. For example, a colony may be disordered. The medium is replaced with fresh medium during the cultivation of the dental pulp suspension to keep the components within certain limits. The medium is replaced, for example, every 1-6 days, 2-4 days, 1 day, 2 days, 3 days or 4 days. All or part of the medium may be replaced with fresh medium. In the case of replacing part of the medium with fresh medium, for example, 1/4 to 4/5, 1/4, 1/3, 1/2 or 4/5 of the amount of the old medium is removed, and then fresh medium is added in an equal amount. Floating cells, pieces of tissue, etc., which were contained in the suspension of dental pulp, can also be removed during this medium exchange.

Cells that have formed a colony in the cell culture plate as a result of culturing the dental pulp suspension can be treated with a protease so that they can be separated from the plate and harvested. The currently used protease is not particularly limited as long as it can degrade protein cell adhesion molecules, but is preferably serine protease, and more preferably trypsin. If trypsin is used, its concentration is preferably 0.1% to 0.5% (w/v) and more preferably 0.25% (w/v).

The cells harvested from the plate described above are suspended in the cell culture medium and then added to the cell culture plate to start culturing in the plate. This is called initial cell culture. The medium used at this time is preferably Dulbecco's modified Eagle's medium to which fetal bovine serum is added. The concentration (v/v%) of fetal bovine serum added to the medium is preferably 8% to 25% and more preferably 8% to 20%, and is, for example, 10%. An example of a detailed composition of Dulbecco's modified Eagle's medium (prior to the addition of fetal calf serum) is shown in Table 1. Accordingly, 3 to 5 mM, eg 4 mM, L-alanyl-L-glutamine can be added to the medium. In addition, if desired, an antibiotic such as streptomycin can be added to the medium. However, usually no antibiotic is added to the medium. In the case where streptomycin is added to the medium, streptomycin is added thereto so that its concentration is from 10 mg/l to 250 mg/l, for example, 100 mg/l. In addition, each component can be replaced by its equivalent, such as its salt.

When starting the initial cell culture described above, cells are added so that the density of living cells in the cell culture plate is preferably 2000 to 30,000 cells/cm ² and more preferably 3000 to 20,000 cells/cm ² , and is, for example, 3000 cells /cm ² , 5000 cells/cm ² , 10,000 cells/cm ² or 20,000 cells/cm ² . The medium is changed during cell culture to keep the components within certain limits. At this time, the medium is replaced, for example, every 1-6 days, 2-4 days, 1 day, 2 days, 3 days or 4 days. All or part of the medium may be replaced with fresh medium. In the case of replacing part of the medium with fresh medium, for example, 1/4 to 4/5, 1/4, 1/3, 1/2 or 4/5 of the amount of the old medium is removed, and then fresh medium is added in an equal amount. Floating cells and the like contained in the original cells are removed during this medium exchange, and as a result, the cells adhering to the cell culture plate are selected.

The initial cell culture is carried out until the cells in the cell culture plate are preferably nearly confluent. For example, culturing is carried out until at least 85%, at least 90%, or at least 95% of the cell culture plate surface on which cells can be attached is covered by cells. Cells that have been cultured until the cells are nearly confluent in the cell culture plate can be treated with a protease so that they can be separated from the plate and harvested. The currently used protease is not particularly limited as long as it can degrade protein cell adhesion molecules, but is preferably serine protease, and more preferably trypsin. If trypsin is used, its concentration is preferably 0.1% to 0.5% (w/v) and more preferably 0.25% (w/v).

After the cells harvested from the above-described plate were resuspended in the cell culture medium, the cells were added to the cell culture plate and then cultured in the plate. The medium used at this time is preferably Dulbecco's modified Eagle's medium to which fetal bovine serum is added. The concentration (v/v%) of fetal bovine serum added to the medium is preferably 8% to 25% and more preferably 8% to 20%, and is, for example, 10%. An example of a detailed composition of Dulbecco's modified Eagle's medium (prior to the addition of fetal calf serum) is shown in Table 1. Accordingly, 3 to 5 mM, eg 4 mM, L-alanyl-L-glutamine may be added to the medium. In addition, if desired, an antibiotic such as streptomycin can be added to the medium. However, usually no antibiotic is added to the medium. In the case where streptomycin is added to the medium, streptomycin is added thereto so that the concentration of streptomycin in the medium is 10 mg/l to 250 mg/l, for example, 100 mg/l. In addition, each component can be replaced by its equivalent, such as its salt. Cell culture is carried out until the cells in the cell culture plate are nearly confluent. For example, culturing is carried out until at least 85%, at least 90%, or at least 95% of the cell culture plate surface on which cells can be attached is covered by cells.

By repeating the collection of cells from the cell culture plate and culturing in the cell culture plate, the number of cells obtained from the dental pulp can be increased. In addition, floating cells are removed by changing the medium at the time of culturing, and cells adhering to the cell culture plate are preferably collected (resultingly selected). Because dental pulp-derived pluripotent stem cells have the ability to attach to a cell culture plate, pluripotent stem cells are enriched by repeating this process. That is, the cells that have gone through the step of culturing the dental pulp suspension in the cell culture plate and the step of harvesting the cells from the plate after culture are dental pulp-derived cells enriched in pluripotent stem cells. By repeating collecting cells from the cell culture plate and culturing cells on it, pluripotent stem cells are further enriched.

Collecting cells from the cell culture plate and culturing cells thereon is preferably repeated 3 to 20 times, more preferably repeated 3 to 8 times, and even more preferably 4 to 8 times, including the original cell culture, and repeated, for example, 4 , 5, 6, 7 or 8 times. Most of the cells that can be obtained by proliferation in a cell culture plate are pluripotent cells derived from the dental pulp, showing a substantially fusiform shape when observed with an optical microscope upon completion of the initial cell culture, but also include other cells. By repeating collecting cells from the cell culture plate and culturing cells on it 3 times, dental pulp-derived pluripotent stem cells are enriched until these cells are predominantly pulp-derived pluripotent cells. When collecting cells from a cell culture plate and culturing cells thereon is repeated 5 or more times, dental pulp-derived pluripotent stem cells are further enriched, and cells other than dental pulp-derived pluripotent cells exhibit essentially spindle-shaped shape when observed with an optical microscope are rarely observed. That is, by repeating collecting cells from the cell culture plate and culturing cells thereon 5 times or more, pluripotent stem cells derived from the dental pulp are essentially isolated.

The proportion of pluripotent stem cells derived from dental pulp in all cells contained in cells thus obtained enriched in pluripotent stem cells derived from dental pulp is preferably greater than or equal to 99%, more preferably greater than or equal to 99.5%, even more preferably greater than or equal to 99.9% and even more preferably greater than or equal to 99.95%. The proportion of pluripotent stem cells derived from dental pulp can be obtained, for example, by dividing the number of cells showing a substantially fusiform shape when observed with an optical microscope by the number of all cells.

Thus obtained dental pulp cells enriched in pluripotent stem cells can be cryopreserved in a state of suspension in a cryopreserving liquid. For cryopreservation, the average cell doubling time in the cell culture plate is preferably within 96 hours, more preferably within 84 hours, even more preferably within 72 hours, even more preferably within 48 hours, and particularly preferably, for example, within 36 hours. or 24 hours. A standard can be set for the mean doubling time, and cells having an average doubling time over a certain time can be cryopreserved and used thereafter. Such a standard may be appropriately set, for example, within 84 hours, 72 hours or 48 hours. Cells that do not meet these standard values can be discarded without cryopreservation to selectively retain only cells capable of undergoing cell division a certain number of times or more. We can say that the shorter the average doubling time of cells, the higher their ability to divide.

The composition of the cryopreservation liquid currently used is not particularly limited, provided that mammalian cells, in particular cells derived from dental pulp enriched in human pluripotent stem cells, can be frozen and thawed without death. The cryopreservation fluid includes a cytoprotective agent. Cytoprotective agents have, for example, the function of inhibiting the formation of ice crystals inside cells and the destruction of cells when cells are frozen. Suitable examples of cytoprotective agents include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin and glycerol. Two or more of these can be combined and used as a cytoprotective agent. In the case of using dimethyl sulfoxide as a cytoprotective agent, its concentration is preferably from 5% to 15% (v/v), and more preferably from 9% to 11% (v/v), and is, for example, 10% (vol./vol.). The cryopreservation fluid may include a buffering agent. A buffering agent that can adjust the pH of the aqueous solution from 6 to 8, for example 6.8 to 7.8, is preferred. Examples of such buffering agents include agents containing carbonate ions, citrate ions and sodium ions. The cryopreservation fluid may further include human serum albumin. In the case of using human serum albumin, its concentration is preferably 40 to 100 g/l, and more preferably 46 to 56 g/l, and may be, for example, 51 g/l. A solution prepared by adding a solution of human serum albumin and dimethyl sulfoxide to Ringer's bicarbonate solution, described below, is a suitable example of a cryopreservation fluid. Both the intermediate cell cryopreservation liquid and the preparation cell cryopreservation liquid to be described below are cryopreservation liquids. In the case of obtaining the intermediate cell freezing step in the production process, the first is called the first cryopreservation liquid (intermediate cell cryopreservation liquid) and the second the second cryopreservation liquid (preparation cell cryopreservation liquid) for convenience.

Cryopreserved dental pulp-derived pluripotent stem cells can be used as a pulp-derived cell preparation, which will be described below, or intermediate cells (which are stored for additional proliferation later). The case of cryopreservation of dental pulp-derived cells enriched in pluripotent stem cells as intermediate cells will be described in detail below.

In the case of cryopreservation of cells derived from dental pulp enriched in pluripotent stem cells as intermediate cells (hereinafter referred to as "the case where cells are frozen as intermediate cells"), collecting the cells from the cell culture plate, and culturing the cells thereon, is repeated preferably 3 to 8 times and more preferably 4 to 8 times, eg 4 times, 5 times, 6 times, 7 times or 8 times, including the original cell culture.

In addition, in the case of freezing cells as intermediate cells, cells collected from a colony formed in the cell culture plate by culturing the dental pulp suspension are cultured in the cell culture plate until the cells are frozen, and it is preferable to cause, at least 10 divisions and more preferably cause at least 15 divisions. For example, cells that have undergone 13-20 divisions, 13-23 divisions, or 14-20 divisions are cryopreserved. Theoretically, the number of cells that have undergone 15 divisions increases by 3 x 10 ⁴ or more times.

In addition, in the case of freezing cells as intermediate cells, the cells usually proliferate until the number of cells obtained from one extracted tooth (for example, the third molar) is preferably 3 × 10 ⁵ or more, more preferably 1 × 10 ⁶ or more, and even more preferably 1 x 10 ⁹ or more.

In addition, for cryopreservation of cells as intermediate cells, the average cell doubling time in the cell culture plate is preferably within 96 hours, more preferably within 84 hours, even more preferably within 72 hours, even more preferably within 48 hours. hours, and particularly preferably, for example, within 24 hours or 36 hours. A standard can be set for the mean doubling time, and cells having an average doubling time over a certain time can be cryopreserved and used thereafter. Such a standard may be appropriately set, for example, within 84 hours, 72 hours or 48 hours. Cells that do not meet these standard values can be discarded without cryopreservation to selectively retain only cells capable of undergoing cell division a certain number of times or more. We can say that the shorter the average doubling time of cells, the higher their ability to divide.

In the case of cell freezing as intermediate cells, the pluripotent stem cell-enriched dental pulp-derived cells that have been harvested and harvested from the cell culture plate by protease treatment are washed with a washing solution before cryopreservation. The wash solution currently used is designed to sufficiently remove the medium and protease, and its composition is not particularly limited. Saline saline, phosphate buffered saline, Ringer's acetate solution, Ringer's bicarbonate solution, and the like can be used as the wash solution, but it is preferable to use a solution prepared by adding human serum albumin to Ringer's bicarbonate solution. Commercially available acetate Ringer's solutions and bicarbonate Ringer's solutions can be used. For example, PLASMA-LYTE (registered trademark) A (Baxter) and Physio (registered trademark) 140 (Otsuka Holdings Co., Ltd.) can be used as Ringer's acetate solutions, and BICARBON (registered trademark) for injection (AJINOMOTO CO., INC.) can be used as ringer's bicarbonate solution. Suitable examples of component composition and electrolyte concentration of Ringer's bicarbonate solution are shown in Tables 2 and 3, respectively.

Table 2. Component composition of ringer's bicarbonate solution

Component mM (range) mm (suitable example) Sodium chloride 94.5–116 105 potassium chloride 3.62–4.42 4.02 Calcium chloride 1.35–1.65 1.5 dihydrochloride magnesium chloride 0.45–0.55 0.5 hexahydrate sodium bicarbonate 22.5–27.5 25 sodium citrate 1.5–1.84 1.67 dihydrate The ratio of the osmotic pressure of the cryopreservative liquid for intermediate cells to physiological saline at pH 6.8–7.8 from 0.9–1.0

Table 3. Electrolyte concentrations in Ringer's bicarbonate solution

Electrolyte meq/l (range) meq/l (suitable example) Na+ 122–149 135 K+ 3.6–4.4 4 Ca2+ 2.7–3.3 3 Mg2+ 0.9–1.1 1 Cl- 102–124 113 HCO3- 22.5–27.5 25 Citrate3- 4.5–5.5 5

In addition, a suitable example of the component composition of a wash solution obtained by adding human serum albumin to Ringer's bicarbonate solution is shown in Table 4. In addition, Table 5 shows a suitable example of the concentration of electrolytes contained in a wash solution obtained by adding human serum albumin to Ringer's bicarbonate solution. Ringer's solution. Sodium acetyltryptophan and sodium caprylate may be omitted from the wash solution components shown in Tables 4 and 5.

Table 4. Component composition of the wash solution

Component mM (range) mm (suitable example) Sodium chloride From 90–110 100 potassium chloride 3.45–4.21 3.83 Calcium chloride 1.29–1.57 1.43 dihydrochloride magnesium chloride 0.429–0.525 0.477 hexahydrate sodium hydrogen 21.4–26.2 23.8 carbonate sodium citrate 1.43–1.75 1.59 dihydrate human serum (10.7–13.1) (11.9) albumen Sodium 0.870–1.06 0.967 acetyltryptophan sodium caprylate 0.874–1.07 0.971

(Note) In the table, the unit of human serum albumin concentration is g/L.

Table 5. The concentration of electrolytes contained in the wash solution (without albumin)

Component mM (range) mm (suitable example) Na+ 117.5–143.6 130.5 K+ 3.45–4.21 3.83 Ca2+ 2.57–3.15 2.86 Mg2+ 0.86–1.05 0.95 Cl- 96.8–118 107.6 HCO3- 21.4–26.2 23, 8 Citrate3- 4.28–5.24 4.76 Acetyltryptophan sodium 0.870–1.06 0.967 sodium caprylate 0.874–1.07 0.971

In the case of freezing the cells as intermediate cells, the cells obtained from the dental pulp enriched in pluripotent stem cells after washing with the washing solution are collected once by a method such as centrifugation. The collected cells are cryopreserved in a state of suspension (intermediate cell suspension). However, an intermediate cell suspension can be prepared by changing the composition of the solution in which the cells are suspended without collecting the cells using an ultrafiltration membrane or the like, and can be cryopreserved. The composition of the solution portion (cryopreservation liquid for intermediate cells) of the intermediate cell suspension is not particularly limited as long as mammalian cells, in particular cells derived from dental pulp enriched in human pluripotent stem cells, can be frozen and thawed without death. Intermediate cell cryopreservation fluid contains a cytoprotective agent. Cytoprotective agents have, for example, the function of inhibiting the formation of ice crystals inside cells and the destruction of cells when cells are frozen. Suitable examples of cytoprotective agents include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin and glycerol. Two or more of these can be combined and used as a cytoprotective agent. In the case of using dimethyl sulfoxide as a cytoprotective agent, its concentration is preferably from 5% to 15% (v/v), and more preferably from 9% to 11% (v/v), and is, for example, 10% (vol./vol.). The cryopreservation fluid may include a buffering agent. A buffering agent that can adjust the pH of the aqueous solution from 6 to 8, for example 6.8 to 7.8, is preferred. Examples of such buffering agents include those containing carbonate ions, bicarbonate ions, citrate ions, and sodium ions. The intermediate cell cryopreservation fluid may further comprise human serum albumin. In the case of using human serum albumin, its concentration is preferably 40 to 100 g/l, and more preferably 46 to 56 g/l, and may be, for example, 51 g/l.

The product obtained by adding a solution of human serum albumin and dimethyl sulfoxide to ringer's bicarbonate solution is a suitable example of a cryopreservation fluid for intermediate cells. Interstitial cell cryopreservation fluid contains 59 to 80.4 mM sodium chloride, 2.3 to 3.08 mM potassium chloride, 0.85 to 1.16 mM calcium chloride dihydrate, 0.28 to 0.385 mM chloride hexahydrate magnesium, 14 to 19.2 mM sodium bicarbonate, 0.94 to 1.28 mM sodium citrate dihydrate, 46 to 56 g/l human serum albumin, 3.73 to 4.55 mM sodium acetyltryptophan, from 3 .74 to 4.58 mM sodium caprylate and 9% to 11% (v/v) DMSO. A suitable example of the composition of the intermediate cell cryopreservation liquid is shown in Table 6. That is, the composition of the intermediate cell cryopreservation liquid consists essentially of 65.8 to 80.4 mM sodium chloride, 2.52 to 3.08 mM potassium chloride, 0 .95 to 1.16 mM calcium chloride dihydrate, 0.315 to 0.385 mM magnesium chloride hexahydrate, 15.7 to 19.2 mM sodium hydrogen carbonate, 1.04 to 1.28 mM sodium citrate dihydrate, 46 to 56 g /L human serum albumin, 3.73 to 4.55 mM sodium acetyltryptophan, 3.74 to 4.58 mM sodium caprylate, and 9% to 11% (v/v) DMSO. Among them, sodium acetyltryptophan and sodium caprylate can be excluded. The ratio of the osmotic pressure of intermediate cell cryopreservation liquid to physiological saline is preferably 0.9 to 1.1.

In addition, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin, and dimethyl sulfoxide can be suitably used as intermediate cell cryopreservation liquid. For example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, in concentrations from 91 to 113 mm, from 2.52 to 3.08 mm, from 0.95 to 1.16 mM, 0.315 to 0.385 mM, 15.6 to 19.2 mM, 1.04 to 1.28 mM, 46 to 56 g/l and 9% to 11% ( v/v) is a suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, in concentrations from 100 to 102 mm, from 2.71 to 2.77 mM, 1.01 to 1.03 mM, 0.335 to 0.345 mM, 16.9 to 17.2 mM, 1.13 to 1.15 mM, 52.2 to 54.3 g/l and from 10.3 to 10.9% (v/v) is another suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, at concentrations of 102 mm, 2.80 mm, 1.05 mm, 0 .35 mM, 17.4 mM, 1.16 mM, 51 g/l and 10% (v/v) is another suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, at concentrations of 101 mm, 2.77 mm, 1.03 mm, 0 .34 mM, 17.2 mM, 1.15 mM, 52 g/l and 10% (v/v) is another suitable example of this. In the case where the intermediate cell cryopreservation liquid further comprises acetyltryptophan or a salt thereof, its concentration is preferably 3.73 to 4.55 mM or 4.24 to 4.41 mM, and is, for example, 4.14 mM or 4.24 mm. In the case where the intermediate cell cryopreservation liquid further comprises caprylic acid or a salt thereof, its concentration is preferably 3.74 to 4.58 mM or 4.25 to 4.43 mM, and is, for example, 4.16 mM or 4.25 mM.

Table 6. Component composition of cryopreservation liquid for intermediate cells

Component mm mm mm mm (Suitable (Suitable (Suitable (Suitable range 1) range 2) example 1) example 2) Sodium chloride 65.8–80.4 70.8–72.3 73.1 72.3 potassium chloride 2.52–3.08 2.71–2.77 2.80 2.77 Calcium chloride dihydrochloride 0.95–1.16 1.01–1.03 1.05 1.03 Magnesium chloride hexahydrate 0.315–0.385 0.335–0.345 0.35 0.34 sodium bicarbonate 15.67–19.15 16.9–17.2 17.41 17.2 Sodium citrate dihydrate 1.04–1.28 1.13–1.15 1.16 1.15 Human serum albumin (46–56) (52.2–54.3) (51) (52.2) Acetyltryptophan sodium 3.73 -4.55 4.24–4.41 4.14 4.24 sodium caprylate 3.74–4.58 4.25–4.43 4.16 4.25 DMSO (9–11) (10.3–10.9) (10) (10)

(Note) The concentration unit of human serum albumin is g/L, the concentration unit of DMSO is % (V/V).

In the case of freezing cells as intermediate cells, the density of cells in suspension in which cells derived from dental pulp enriched in pluripotent stem cells are suspended in the intermediate cell cryopreservation liquid is not particularly limited, but is preferably 2 x 10 ⁶ to 8 x 10 ⁷ cells/ml, more preferably from 4 x 10 ⁶ to 3 x 10 ⁷ cells/ml, and even more preferably from 8 x 10 ⁶ to 2 x 10 ⁷ cells/ml, and is, for example, 1.1 x 5 10 ⁷ cells/ml. Dental pulp-derived cells enriched in pluripotent stem cells suspended in intermediate cell cryopreservation fluid are placed in a cell cryopreservation container and then cryopreserved.

Meanwhile, the amount of the cell suspension to be dispensed in one cell cryopreservation container needs to be appropriately adjusted depending on the application and the like, and is preferably 1 to 20 ml. Further, the number of cells to be placed in one cell cryopreservation container is preferably 5 x 10 ⁶ to 9.2 x 10 ⁸ .

The container used as the cell cryopreservation container is not particularly limited as long as the material exhibits durability at low temperatures and the container is not damaged even if its contents are frozen and thawed. Commercially available containers can also be used as cryopreservation containers. For example, glass and plastic (eg, polyolefin resin) cryotubes can be suitably used as containers for cell cryopreservation. In this case, a vial whose material is borosilicate glass in particular can be suitably used as a glass container. In addition, herein, the polyolefin resin includes polyethylene, high-density polyethylene, low-density polyethylene, polypropylene, an ethylene-propylene copolymer, and a thermoplastic elastomer. Cell freezing is preferably performed by distributing the cell suspension into a cell cryopreservation container, followed by a gradual decrease in temperature. For example, a method of lowering the temperature at a rate of 1°C per minute to -6°C and then rapidly freezing the cells can be used as a cell freezing method. Cells are stored frozen. The temperature at which the cells are cryopreserved is preferably less than or equal to -130°C, more preferably less than or equal to -170°C, and even more preferably less than or equal to -180°C. For example, the cells may be stored in a state in which the cell cryopreservation container is immersed in liquid nitrogen (boiling point: -196°C).

Such cells, cryopreserved as intermediate cells, are thawed at use for use in the next step in obtaining a cell preparation derived from dental pulp.

The provision of a step to temporarily retain proliferated cells as intermediate cells in the process of obtaining pluripotent stem cell-enriched dental pulp-derived cells is extremely important from the point of view of controlling the process of pulp-derived pluripotent stem cell-enriched cells. For example, in the case where the desired number of extracted teeth cannot be obtained at one time, if the cultivation of cells obtained from the pulp of the tooth is started at any time using the obtained extracted teeth as raw materials for the production of end products, the batch sizes of the end products are small sizes, and batch management becomes difficult. However, if intermediate cells are produced and stored, once the intermediate cells are accumulated to a certain number, the next production process can be started with these intermediate cells as one batch. In this way, the batch size of finished products can be increased, and batch management becomes easier.

In addition, by providing the step of maintaining the cells as intermediate cells, quality tests for properties and the like can be performed. cells at an intermediate stage of the manufacturing process. Accordingly, only when the cells obtained as intermediate cells meet the desired quality standards, the cells can be used for the next production process. That is, cells that do not meet the desired quality standards during, before, or after completion of the intermediate cell manufacturing process can be excluded. Accordingly, cells that do not meet the desired quality standards are not transferred to subsequent stages. Therefore, it is possible to increase the likelihood of producing end products that meet the desired quality standards. That is, you can increase the yield at the time of production and reduce production costs.

Cryopreservation of cells during production is usually associated with the risk of changing cell properties before and after cell freezing. In addition, the ability of cells to proliferate sometimes deteriorates after cryopreservation. On the contrary, in the cells obtained as intermediate cells of the present invention, neither a change in the properties of cells before and after freezing, nor a deterioration in the ability to proliferate after cryopreservation is recognized.

Dental pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells preferably have a high proportion of living cells contained within the cells. The proportion (cell viability) of living cells before cryopreservation is preferably greater than or equal to 50%, more preferably greater than or equal to 60%, and even more preferably greater than or equal to 70%, and preferably, for example, greater than or equal to 80%, greater than or equal to 90% or greater than or equal to 95%. It is also possible to retain only cells having a certain or greater cell viability before cryopreservation as intermediate cells in accordance with appropriately established standards.

Dental pulp-derived cells enriched in pluripotent stem cells to be retained as intermediate cells preferably undergo at least 10 cell divisions in an in vitro medium. For example, cells undergo at least 15, 16, or 17 cell divisions. The average cell division time during the cell division period is preferably 48 hours.

In the present invention, inducing cell division in an in vitro medium means that, for example, cell division occurs in a state where cells are added to a culture container such as a cell culture flask along with the cell culture medium. The cultivation temperature at this time is preferably 34°C to 38°C, and more preferably 36°C to 38°C, and is, for example, 37°C. In addition, the cells are preferably cultured in a humid environment in the presence of 5% CO2. The cell culture medium currently used is not particularly limited as long as it can be used for mammalian cell culture, and is, for example, Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS). The concentration of FBS contained in the medium is preferably 2% to 20%, more preferably 5% to 20%, and even more preferably 8% to 15%, for example 10%. In addition, the concentration of glucose in DMEM is from 5 to 20 mm, for example, 5 mm. Examples of suitable culture conditions include a culture temperature of 37° C., a moist medium in the presence of 5% CO2 and DMEM containing about 5 mM glucose and 10% FBS as medium. Method for culturing cells obtained from dental pulp as described in Example 5 (i.e., in cell culture plate, DMEM medium containing 5.56 mM glucose and to which 10% FBS has been added, in the presence of 5% CO2 and 37°C) is an example of a suitable method for inducing cell division in an in vitro environment.

Cell subculture, in the case where cells are dividing in an in vitro medium, is performed such that the cells are separated from the culture container and then seeded into a new culture container so that 1/20 to 1/3 of the bottom of the culture container is covered cells. In the case where at least 70%, at least 80%, or at least 90% of the bottom of the culture container is covered with cells due to cell division, subculture is repeated.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes, an increase in aggrecan expression is generally observed. . In addition, when dental pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are cultured in a medium containing a substance known to induce differentiation into osteocytes, an increase in the amount accumulated in the cells is generally observed. calcium. That is, dental pulp-derived cells enriched in pluripotent stem cells have the ability to differentiate into chondrocytes and osteocytes when observed as a whole.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed as a whole, the cells are positive for at least one of CD73, CD90, CD105, and CD166, and negative for at least one of CD34 and CD45 in the surface antigen marker expression patterns. For example, when cells are observed as a whole, cells are positive for CD73 and CD90 and negative for CD34 in surface antigen marker expression patterns. In addition, for example, when the cells are observed as a whole, the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in surface antigen marker expression patterns.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, these cells, for example, are negative for at least one of CD40, CD80, CD86, and MHC class II antigen in expression patterns of surface antigen markers. For example, when the cells are observed as a whole, the cells are negative for CD40 antigen, CD80, CD86 and MHC class II in surface antigen marker expression patterns. It is known that when cells positive for these surface antigen markers are transplanted into allogeneic individuals, the cells tend to be recognized as an antigen to be eliminated from the living body. In the case where the cells are negative for at least one of these surface antigen markers, this means that the cells derived from the dental pulp, enriched in pluripotent stem cells, which should be preserved as intermediate cells, have the property of low immunogenicity up to to such an extent and are unlikely to be excluded from a living organism when transplanted into allogeneic individuals. In addition, cells can remain negative for CD40, CD80 and CD86 and be positive for the MHC class II antigen in surface antigen marker expression patterns when the cells are stimulated with IFN-γ. For example, cells remain negative for CD40, CD80, and CD86 and are positive for MHC class II antigen in surface antigen marker expression patterns when cells are stimulated with IFN-γ.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, these cells are, for example, positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen in expression patterns of surface antigen markers. In addition, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ.

In one embodiment, regarding characteristic (b-1), when the cells derived from the dental pulp, enriched in pluripotent stem cells, which should be preserved as intermediate cells, are observed in general, these cells are positive in at least one, preferably all of CD47, CD81, CD90, CD147 and HLA-A, -B and -C in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding the characteristic (b-2), when cells derived from the dental pulp, enriched with pluripotent stem cells, which should be preserved as intermediate cells, are observed in general, the cells are positive for at least one , preferably for all of CD29, CD46, CD55, CD59, CD73 and CD140b in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (b-3), when cells derived from the dental pulp, enriched in pluripotent stem cells, which should be preserved as intermediate cells, are observed in general, the cells are positive in at least one , preferably all of CD9, CD44, CD49b, CD49c, CD98 and EGF-R in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 65%, more preferably at least 75%, and even more preferably at least 80% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (b-4), when cells derived from dental pulp enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, the cells are positive for at least one , preferably for all of CD49f and CD166 in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 70%, and even more preferably at least 75% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (b-5), when cells derived from the dental pulp, enriched in pluripotent stem cells, which should be preserved as intermediate cells, are observed in general, the cells are positive for at least one , preferably all of CD10, CD13, CD58, CD63, CD105, CD151 and CD164 in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 65%, and even more preferably at least 70% of the observed cells are positive for these antigens.

In one embodiment, the dental pulp-derived cells enriched in pluripotent stem cells to be retained as intermediate cells preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in those described above (b -1), (b-2), (b-3), (b-4) and (b-5). For example, cells having the characteristics shown in (b-1) and (b-2) described above are a suitable embodiment of the present invention.

In one embodiment, regarding characteristic (b-6), when the cells derived from the dental pulp, enriched with pluripotent stem cells, which should be preserved as intermediate cells, are observed in general, these cells are negative, at least one, preferably all of CD120b, CD132, CD158a, CD161, CD184, CD195, CD206, CD210, CD212, CD226, CD244, CD267, CD278, CD279, CD282, CD294, NKB1, SSEA-1, TRA-1-60, TRA -1-81, Vp23, SSEA-3, CLA and β7 integrin in expression patterns of surface antigen markers.

Herein, when individual cells included in all cells are observed, preferably no more than 10%, more preferably no more than 5%, and even more preferably no more than 2%, and even more preferably no more than 1% of the observed cells are positive for these antigens.

In one embodiment, with respect to characteristic (b-7), when cells derived from dental pulp enriched in pluripotent stem cells to be preserved as intermediate cells are observed as a whole, the cells are negative in at least one, preferably for all of CD8b, CD11b, CD15, CD16, CD19, CD24, CD31, CD32, CD62E, CD62P, CD66f, CD86, CD88, CD94, CD100, CD103, CD104, CD114, CD117, CD118, CD121b, CD122, CD123, CD124, CD126, CD127, CD128b, CD135, CD137, CD137 ligand, CD150, CD163, CD172b, CD177, CD178, CD180, CD197, CD220, CD229, CD231, CD255, CD268, CD305, CD314, CD321, CDw3327, CD w3327, CD7 , CD336, BLTR-1, CLIP, CMRF-44, CMRF-56, fMLP-R, Vβ8, invariant NKT, and γδ TCR in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably no more than 10%, more preferably no more than 5%, and even more preferably no more than 2% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (b-8), when the cells derived from the dental pulp, enriched with pluripotent stem cells, to be maintained as intermediate cells, observed in general, the cells are negative in at least one, preferably in all of: CD1a, CD1b, CD1d, CD2, CD3, CD5, CD6, CD7, CD8a, CD11c, CD15, CD18, CD21, CD22, CD23, CD25, CD26, CD27, CD28, CD33, CD34, CD35, CD37, CD38, CD40, CD41a, CD41b, CD42b, CD45, CD45RB, CD45RO, CD48, CD50, CD53, CD62L, CD64, CD66 (a, c, d, e), CD69, CD70, CD72, CD74, CD80, CD84, CD85 , CD87, CD89, CDw93, CD97, CD106, CD134, CD138, CD141, CD144, CD154, CD158b, CD162, CD183, CD205, CD235a, CD271, CD309, CD326, CD337, αβ TCR and MHC class II antigen in expression patterns surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably only at most 10% and more preferably only at least 5% of the observed cells are positive for these antigens.

In one embodiment, the dental pulp-derived cells enriched in pluripotent stem cells to be retained as intermediate cells preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in (b- 1), (b-2), (b-3), (b-4), (b-5), (b-6), (b-7) and (b-8). For example, cells having the characteristics shown in (b-1) and (b-6) described above are a suitable embodiment of the present invention.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, these cells express, for example, prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF ), and the expression level of prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are cultured in the presence of IFN-γ, the amount of kynurenine secreted increases.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed as a whole, the cells are positive for at least one or all of CD47, CD81, and CD147 and negative for at least one or all of CD19, CD34 and CD206 in surface antigen marker expression patterns. In addition, in another embodiment, the cells are positive for at least one or all of CD47, CD81 and CD147 and negative for at least one or all of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a and SSEA-1 in expression patterns of surface antigen markers.

In one embodiment of the present invention, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, these cells are, for example, positive for at least one of CD73, CD90, CD105 and CD166 and negative for at least one of CD34 and CD45 in surface antigen marker expression patterns.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen in expression patterns of surface antigen markers. Thus, for example, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ. In addition, cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and the amount of secreted prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells.

In one embodiment, when pulp-derived cells enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, the expression level of aggrecan is increased when the cells are cultured in a medium containing a substance known to , induces differentiation into chondrocytes. In addition, in the case where cells are cultured in a medium containing a substance known to induce differentiation into osteocytes, the amount of calcium accumulated in the cells is increased. That is, dental pulp-derived cells enriched in pluripotent stem cells have the ability to differentiate into chondrocytes and osteocytes when observed as a whole.

A method in which only cells showing certain expression patterns are retained as intermediate cells in accordance with appropriately established standards based on the above-described expression patterns of surface antigen markers shown by dental pulp-derived cells enriched in pluripotent stem cells of the present invention and/ or other gene expression patterns can also be used to control the manufacturing process.

Dental pulp-derived cells enriched in pluripotent stem cells to be retained as intermediate cells can mainly be pluripotent stem cells (dental pulp-derived pluripotent stem cells) derived from dental pulp. Under an optical microscope, they are observed as attached cells of essentially spindle shape in a flat culture, and the proportion of essentially spindle cells in all cells is preferably greater than or equal to 99%, more preferably greater than or equal to 99.5%, even more preferably greater than or equal to 99, 9% and even more preferably greater than or equal to 99.95%. The proportion of pluripotent stem cells derived from dental pulp can be obtained by dividing the number of cells showing a substantially fusiform shape when observed with an optical microscope by the number of all cells. A method can also be used to control the manufacturing process, in which only groups of cells having a certain value or more than a proportion of essentially spindle cells are stored as intermediate cells in accordance with properly established standards based on the foregoing.

In one embodiment, when cells derived from dental pulp enriched in pluripotent stem cells to be preserved as intermediate cells are observed as a whole, the cells preferably have the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes. That is, in the case where intermediate cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes, the expression level of aggrecan is increased. In addition, when intermediate cells are observed as a whole, in the case where the cells are cultured in a medium containing a substance known to induce differentiation into osteocytes, the amount of calcium accumulated in the cells increases. The fact that intermediate cells have the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes can be investigated, respectively, using the methods described in examples 20 and 19. these methods can be considered as intermediate cells to be preserved. However, methods for testing the ability to differentiate are not limited to the methods described in Examples 20 and 19. Only groups of cells found to be able to differentiate into chondrocytes and the ability to differentiate into osteocytes by other suitable methods can also be considered as intermediate cells to be preserved.

In one embodiment, the pulp-derived cells enriched in pluripotent stem cells to be retained as intermediate cells may be the same or substantially the same as the intermediate cells used in a further culture step, which will be detailed described below.

The properties of cells cryopreserved as intermediate cells are preferably preserved before and after thawing. That is, when dental pulp-derived cells enriched in pluripotent stem cells that have been cryopreserved as intermediate cells are thawed, viability, surface antigen marker expression patterns, cell morphology, and cell differentiation ability are preferably substantially the same as cells before thawing. Only cells with properties preserved before and after thawing can be used for the subsequent culture step as intermediate cells by checking the properties of the intermediate cells after thawing or in culturing after thawing.

The viability of the intermediate cells to be used in the further culture step after thawing is preferably greater than or equal to 50%, more preferably greater than or equal to 60%, and even more preferably greater than or equal to 70%. %, and preferably is, for example, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95%. Only intermediate cells having a certain value or greater viability after thawing may be used for the subsequent culture step in accordance with appropriately established standards. Cell viability after thawing is essentially 80% to 98% or 85% to 95%.

When the cells cryopreserved as intermediate cells are cultured after thawing, the cells preferably have the ability to undergo at least 10 cell divisions, and more preferably have the ability to undergo at least 15 cell divisions and have, for example, the ability to undergo at least 14 cell divisions. In addition, when the cells frozen as intermediate cells are cultured after thawing, the average doubling time of the cells in the cell culture plate is preferably within 96 hours, more preferably within 84 hours, even more preferably 72 hours, even more preferably 48 hours. . and even more preferably within 36 hours. For example, standards for cell division ability and mean doubling time can be set, and only cells that have the ability to cell divide a certain number of times or more, and of which the mean doubling time is within a certain time, can be used for subsequent applications. Such standards can be set accordingly. For example, cells having the ability to undergo at least 10 cell divisions and having an average doubling time of 96 hours, cells having the ability to undergo at least 14 cell divisions and having an average doubling time of 72 hours, and cells having the ability to undergo at least 15 cell divisions and having an average doubling time of 72 hours. Only intermediate cells having the ability to undergo cell divisions a certain number of times or more can be used for the subsequent culture step without using cells that do not meet these standards. Culture conditions at this stage may be the same or equivalent to those described above for cells derived from dental pulp enriched in pluripotent stem cells.

Cells to be cryopreserved as intermediate cells preferably have undergone at least 10 cell divisions in in vitro medium. For example, cells undergo at least 15, 16, or 17 cell divisions. The average cell division time during the cell division period is preferably 48 hours.

When intermediate cells to be used in a further culture step are cultured in a medium containing a substance known to induce differentiation into chondrocytes, an increase in the level of expression of aggrecan as a whole is observed. In addition, when the pulp-derived cells of the present invention enriched in pluripotent stem cells are cultured in a medium containing a substance known to induce differentiation into osteocytes, an increase in the amount of calcium accumulated in the cells is generally observed. Cells have the ability to differentiate into chondrocytes and osteocytes when observed as a whole.

In one embodiment, when intermediate cells to be used in a further culturing step are observed as a whole, the cells are positive for at least one of CD73, CD90, CD105, and CD166 and negative for at least one of CD34 and CD45 in expression patterns of surface antigen markers after cell thawing or during cell culture after thawing. For example, cells are positive for CD73 and CD90 and negative for CD34. In addition, for example, the cells are positive for CD73 and CD90 and negative for CD34. The cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45.

In addition, in one embodiment, when intermediate cells to be used for a further culturing step are observed as a whole, the cells are negative for at least one of the CD40, CD80, CD86 and MHC class II antigens in surface marker expression patterns. antigens immediately after thawing of cells or when culturing cells after thawing. For example, cells negative for CD40, CD80, CD86 and MHC-class II antigen. It is known that when cells positive for these surface antigen markers are transplanted into allogeneic individuals, the cells tend to be recognized as an antigen to be eliminated from the living body. In the case where the cells are negative for these surface antigen markers, this means that the intermediate cells to be used for the next culturing step are hypoimmunogenic to that extent and tend to be more difficult to be eliminated from the living organism when transplanted into allogeneic individuals. In addition, cells can remain negative for CD40, CD80 and CD86 and be positive for the MHC class II antigen in surface antigen marker expression patterns when the cells are stimulated with IFN-γ. For example, cells remain negative for CD40, CD80, and CD86 and are positive for MHC class II antigen in surface antigen marker expression patterns when cells are stimulated with IFN-γ.

In one embodiment, when intermediate cells to be used in a further culturing step are observed in general immediately after thawing or in culturing after thawing, then these cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen in expression patterns of surface antigen markers. In addition, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ.

In one embodiment, regarding characteristic (c-1), when the intermediate cells to be used for the further culturing step are observed as a whole, the cells are positive for at least one, preferably for all of CD47, CD81, CD90, CD147 and HLA-A, -B and -C in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the observed cells are positive for these antigens .

In one embodiment, regarding characteristic (c-2), when intermediate cells to be used in a further culture step are observed in general, the cells are positive for at least one, preferably for all of CD29, CD46, CD55, CD59, CD73 and CD140b in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the observed cells are positive for these antigens .

In one embodiment, regarding characteristic (c-3), when the intermediate cells to be used for the further culturing step are observed as a whole, the cells are positive for at least one, preferably for all of CD9, CD44, CD49b, CD49c, CD98 and EGF-R in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 65%, more preferably at least 75%, and even more preferably at least 80% of the observed cells are positive for these antigens .

In one embodiment, regarding characteristic (c-4), when intermediate cells to be used in a further culturing step are observed in general, the cells are positive for at least one, preferably for all of CD49f and CD166 in expression patterns surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 70%, and even more preferably at least 75% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (c-5), when cells derived from dental pulp enriched in pluripotent stem cells to be preserved as intermediate cells are observed in general, the cells are positive for at least one, preferably all, of CD10, CD13, CD58, CD63, CD105, CD151 and CD164 in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 65%, and even more preferably at least 70% of the observed cells are positive for these antigens.

In one embodiment, intermediate cells to be used in a further culture step preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in (c-1), (c-2), ( c-3), (a-4) and (c-5). For example, cells having the characteristics shown in (c-1) and (c-2) above are a suitable embodiment of the present invention.

In one embodiment, regarding characteristic (c-6), when the intermediate cells to be used for the further culturing step are observed as a whole, the cells are negative for at least one, preferably all of CD120b, CD132, CD158a, CD161, CD184, CD195, CD206, CD210, CD212, CD226, CD244, CD267, CD278, CD279, CD282, CD294, NKB1, SSEA-1, TRA-1-60, TRA-1-81, Vp23, SSEA-3, CLA and β7 integrin in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably no more than 10%, more preferably no more than 5%, even more preferably no more than 2%, and even more preferably no more than 1% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (c-7), when the intermediate cells to be used for the further culturing step are observed as a whole, the cells are negative for at least one, preferably all of CD8b, CD11b, CD15, CD16, CD19, CD24, CD31, CD32, CD62E, CD62P, CD66f, CD86, CD88, CD94, CD100, CD103, CD104, CD114, CD117, CD118, CD121b, CD122, CD123, CD124, CD126, CD127, CD128b, CD135, CD137, CD137, CD150, CD163, CD172b, CD177, CD178, CD180, CD197, CD220, CD229, CD231, CD255, CD268, CD305, CD314, CD321, CDw327, CDw328, CD329, CD335, CD335, CD1336 CLIP, CMRF- 44, CMRF-56, fMLP-R, Vβ8, invariant NKT and γδ TCR in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at most 10%, more preferably at most 5%, and even more preferably at most 2% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (c-8), when intermediate cells to be used for a further culturing step are observed in general, the cells are negative for at least one, preferably for all of CD1a, CD1b, CD1d, CD2 , CD3, CD5, CD6, CD7, CD8a, CD11c, CD15, CD18, CD21, CD22, CD23, CD25, CD26, CD27, CD28, CD33, CD34, CD35, CD37, CD38, CD40, CD41a, CD41b, CD42b, CD45 , CD45RB, CD45RO, CD48, CD50, CD53, CD62L, CD64, CD66 (a, c, d, e), CD69, CD70, CD72, CD74, CD80, CD84, CD85, CD87, CD89, CDw93, CD97, CD106, CD134, CD138, CD141, CD144, CD154, CD158b, CD162, CD183, CD205, CD235a, CD271, CD309, CD326, CD337, αβ TCR and MHC class II antigen in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at most 10% and more preferably at most 5% of the observed cells are positive for these antigens.

In one embodiment, intermediate cells to be used in a further culture step preferably have two or more, more preferably three or more, and even more preferably all of the characteristics shown in (c-1), (c-2), ( c-3), (a-4), (c-5), (c-6), (c-7) and (c-8). For example, cells having the characteristics shown in (c-1) and (c-6) described above are a suitable embodiment of the present invention.

In addition, when intermediate cells to be used in a further culture step are observed in general, the cells express, for example, prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and the level of expression of prostaglandin E2 (PGE2) increases beyond by stimulating TNF-α cells.

In one embodiment of the present invention, when intermediate cells to be used in a further culture step are cultured in the presence of IFN-γ, the amount of kynurenine secreted increases.

In one embodiment of the present invention, when intermediate cells to be used in a further culturing step are observed as a whole, the cells are positive for at least one or all of CD47, CD81 and CD147 and negative for at least one or all of CD19, CD34 and CD206 in surface antigen marker expression patterns. Further, in one embodiment of the present invention, when pluripotent stem cell-enriched dental pulp-derived cells are observed in general, these cells are positive for at least one or all of CD47, CD81, and CD147 and negative for at least one or all of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a, and SSEA-1 in surface antigen marker expression patterns.

In one embodiment, when intermediate cells to be used in a further culturing step are observed as a whole, the cells are negative for at least one, e.g., all of CD19, CD26, CD106, CD117, and CD271 in surface marker expression patterns. antigens.

In one embodiment, when intermediate cells to be used in a further culturing step are observed as a whole, the cells are positive for at least one, for example, for all CD140b and HLA-A, -B and -C in marker expression patterns surface antigens.

In one embodiment of the present invention, when intermediate cells to be used for a further culturing step are observed as a whole, the cells are positive for at least one, for example, for all of CD46, CD47, CD55, CD58 and CD59 in patterns expression of surface antigen markers.

In one embodiment of the present invention, when intermediate cells to be used for a further culture step are observed as a whole, the cells are, for example, positive for at least one of CD73, CD90, CD105 and CD166. and negative for at least one of CD34 and CD45 in surface antigen marker expression patterns.

In addition, in one embodiment, when intermediate cells to be used for a further culture step are observed in general, the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC- class II in expression patterns of surface antigen markers. At this time, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ. In addition, cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and secreted prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells. In addition, the amount of secreted kynurenine is increased by stimulation of IFN-γ cells.

When cells contained in dental pulp-derived cell preparations are cultured, the cells secrete various humoral factors.

In one embodiment, regarding characteristic (c-9), when cells contained in cell preparations derived from dental pulp are cultured, the cells express at least one, preferably all, of MMP-2, IGFBP-4, and cystatin C .

In one embodiment, regarding characteristic (c-10), when intermediate cells are cultured to be used in a further culture step, the cells express at least one, preferably all of IL-6, IL-11, MCP-1, IL-8, GROα, HGF, VEGF, VCAM-1, TIMP-3, TIMP-2 and TIMP-1.

In addition, regarding characteristic (c-11), in one embodiment, when intermediate cells to be used in a further culture step are cultured, the cells express at least one, preferably all of IL-23, TNF-α, IL -18, IL-33, IL-27, TARC, ENA-78, MIP-3α, MIP-1β, IP-10, SCF and ICAM-1.

In addition, with respect to characteristic (c-12), in one embodiment, when intermediate cells are cultured to be used for further culture steps, the cells do not express or almost do not express at least one, preferably all of IL-21, IFN-a, eotaxin, MIP-1a, MIG, I-TAC and GM-CSF.

In addition, regarding the characteristic (c-13), in one embodiment, in the case where the intermediate cells to be used for the further stage of cultivation are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-6 is higher than in the case of cell culture in the absence of such.

In addition, regarding the characteristic (c-14), in one embodiment, in the case where the intermediate cells to be used for the further culture step are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-11 is higher than in case of cell culture in the absence of such.

In addition, regarding the characteristic (c-15), in one embodiment, in the case where the intermediate cells to be used for the further culture step are cultured in the presence of TNF-α or IFN-γ, the expression level of IP-10 is higher than in case of cell culture in the absence of such.

In addition, regarding the characteristic (c-16), in one embodiment, in the case where the intermediate cells to be used for the further stage of cultivation are cultured in the presence of TNF-α or IFN-γ, the expression level of MCP-1 is higher than in case of cell culture in the absence of such.

In addition, with respect to characteristic (c-17), in one embodiment, GM-CSF expression is induced when intermediate cells to be used in a further culture step are cultured in the presence of TNF-α, but GM-CSF expression is not induced when the cells are cultured. in the presence of IFN-γ.

In addition, with respect to characteristic (c-18), in one embodiment, the expression level of HGF is reduced when intermediate cells to be used in a further culture step are cultured in the presence of TNF-α compared to when the cells are cultured in its absence, when In this case, the expression level of HGF is increased when cells are cultured in the presence of IFN-γ compared to when cells are cultured in its absence.

In addition, with respect to characteristic (c-19), in one embodiment, in the case where the intermediate cells to be used for the further culturing step are cultured in the presence of TNF-α, the expression level of IL-8 is greater than when the cells cultivated in his absence.

In one embodiment, intermediate cells to be used in a further culture step preferably have two or more, more preferably three or more, even more preferably four or more, and even more preferably all the characteristics shown in the above: described with (c- 9) to (c-19). For example, cells having the characteristics shown in (c-9) and (a-15) described above and cells having the characteristics shown in (c-9), (c-14) and (c-15) described above represent a suitable embodiment of the present invention.

Furthermore, when intermediate cells to be used for the next culturing step are observed as a whole immediately after thawing or in culturing after thawing, the cells have the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes. That is, the expression level of aggrecan increases when cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes, and the amount of calcium accumulated in cells increases when cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes. is known to induce differentiation into osteocytes. The fact that intermediate cells have the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes can be investigated, respectively, using the methods described in examples 20 and 19. Only groups of cells that show the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes using of these methods, can be considered as intermediate cells that will be used for a further stage of cultivation. However, methods for testing these differentiation abilities are not limited to the methods described in the examples. Only groups of cells having the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes by other suitable methods can also be used as intermediate cells in the subsequent culture step.

A method in which only cells showing specific expression patterns are considered as intermediate cells to be used in a further culture step in accordance with appropriately established standards based on the above-described expression patterns of surface antigen markers presented by cells derived from dental pulp, enriched in pluripotent stem cells according to the present invention and/or other gene expression patterns can also be used to control the manufacturing process.

In addition, when intermediate cells to be used in a further culturing step are observed in general immediately after thawing or in culturing after thawing, these cells mainly refer to dental pulp-derived pluripotent stem cells. Under an optical microscope, they are observed as attached cells of essentially spindle shape in flat culture, and the proportion of essentially spindle cells in all cells is preferably greater than or equal to 99%, more preferably greater than or equal to 99.5%, even more preferably greater than or equal to 99, 9% and even more preferably greater than or equal to 99.95%. Only groups of cells that have a certain value or a large proportion of essentially spindle cells can be used as intermediate cells in the subsequent stage of cultivation in accordance with properly established standards based on the foregoing.

In addition, when intermediate cells to be used for a further culturing step are cultured after thawing, the cells preferably have the ability to undergo at least 10 cell divisions, and more preferably have the ability to undergo at least 15 cell divisions, and have, for example, the ability to undergo at least 14 cell divisions. In addition, the average doubling time of the intermediate cells to be used in the further culture step when they are thawed and cultured in the cell culture plate is preferably within 96 hours, more preferably within 84 hours, even more preferably within 72 hours, still more preferably within 48 hours and even more preferably within 36 hours. At this point, cell culture conditions that are the same or equivalent to those of the above-described pluripotent stem cell-enriched dental pulp-derived cells can be used, for example, conditions (i.e., in cell culture plate, in DMEM medium that contains 5 ,56 mM glucose and to which 10% FBS is added, in the presence of 5% CO2 and 37°C) described in Example 5. Only groups of cells that have the ability to undergo cell divisions a certain number of times or more can be used as intermediate cells for subsequent cultivation step in accordance with appropriately established standards based on the foregoing. In addition, only groups of cells having an average doubling time within a certain time can be used as intermediate cells in a subsequent culture step in accordance with properly established standards. Such standards are, for example, cells capable of undergoing at least 10 cell divisions that have an average doubling time of 96 hours, cells that have the ability to undergo at least 14 cell divisions that have an average doubling time of 72 hours, and cells having the ability to undergo at least 15 cell divisions, which have an average doubling time of 72 hours.

That is, the intermediate cells to be used in the further culturing step retain the properties of stem cells that are highly dividing and can be differentiated into two or more types of cells. Intermediate cells to be used in a further culture step may also be referred to as cells having a high colony-forming capacity. As used herein, colony-forming ability refers to the ability of dividing cells to form colonies on a plate when the cells are plated and cultured.

When the intermediate cells are thawed, the average cell diameter in the state in which the cells are made to float in a solution, such as a medium, is preferably less than or equal to 20 μm or less than or equal to 18 μm, and is, for example, 10 to 20 μm, 10 up to 18 µm, 12 to 20 µm, 14 to 20 µm and 16 to 20 µm.

Quality test items conducted during, before or after completion of the intermediate cell production process are presented below as examples of quality tests a to k. Quality tests are conducted on one or more items. All of these items can be done. The cells to be used for quality tests at this point are either cells (1), as intermediate cells, obtained by fractionating some cells before they are suspended in cryopreservation liquid, or cells (2), as intermediate cells, obtained by fractionation of some cells before being suspended in cryopreservation fluid and further subculture of these fractionated cells, cells (3), as intermediate cells, before freezing, which were suspended in cryopreservation fluid for freezing, cells (4) as intermediate cells, immediately after thawing frozen cells, or cells (5) as intermediate cells, obtained by thawing frozen cells and then culturing the thawed cells. Unless otherwise noted, similar quality tests may be performed on two or more of these cells (1) - (5).

(Quality test a) Checking that the proportion of the number of cells showing a substantially fusiform shape in all cells when observed under an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9%, or greater than or equal to 99.95,

(Quality test b) Checking that the cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95%,

(Quality test c) Checking that cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression patterns, or checking that cells are positive for at least one of CD73 and CD90 and CD34 negative in expression patterns of cell surface antigen markers (Quality test d) Confirmation that cells are negative for CD40, CD80, CD86 and MHC class II antigen in expression patterns of cell surface antigen markers, or checking that cells are negative for at least CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression patterns,

(Quality test e) Checking that cells remain negative for CD40, CD80 and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression patterns when cells are stimulated with IFN-γ, or checking at least one of cells being CD40 negative, CD80 negative, CD86 negative, and MHC class II antigen positive in cell surface antigen marker expression patterns when cells are stimulated with IFN-γ (Quality test f) Checking that cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and the expression level of prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells,

(Quality test g) Checking that the expression level of aggrecan is increased when cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes,

(Quality test h) Checking that the amount of calcium accumulated in cells is increased when cells are cultured in a medium containing a substance known to induce differentiation into osteocytes,

(Quality test i) Checking that the cells have the ability to undergo at least 10, 14 or 15 cell divisions in a cell culture plate,

(Quality test j) Checking that the average cell doubling time in the cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours, or 36 hours during the quality test period i.

(Quality check k) Checking that the average cell diameter in the state where cells float in the medium is less than or equal to 20 µm, less than or equal to 18 µm, 10 to 20 µm, 10 to 20 µm, 10 to 18 µm , 12 to 20 µm, 14 to 20 µm, or 16 to 20 µm.

The above quality tests can be carried out on 10 items (Quality test a) - (Quality test j). In addition, random 9 points can be selected from these 10 points for quality tests. In the case where 9 items are selected for quality tests, for example, quality tests a, b, c, d, e, f, g, i, and j can be selected, without limitation. In addition, random 8 points can be selected from these 10 points for quality tests. In the case where 8 tests are selected to conduct quality tests, for example, quality tests a, b, c, d, e, f, i, and j may be selected, without limitation. In addition, random 7 points can be selected from these 10 points for quality tests. In the case where 7 items are selected for performing quality tests, for example, quality tests a, b, c, d, e, i, and j may be selected, without limitation. In addition, random 6 points can be selected from these 10 points for quality tests. In case 6 items are selected for quality tests, for example, quality tests a, b, c, d, i and j can be selected, without being limited to the above. In addition, random 5 points can be selected from these 10 points for quality tests. In the case where 5 items are selected for performing quality tests, for example, quality tests a, b, c, i, and j can be selected without limitation. In addition, random 4 points can be selected from these 10 points for quality tests. In the case where 4 items are selected for performing quality tests, for example, quality tests a, b, c, and j can be selected, without limitation. In addition, random 3 elements can be selected from these 10 elements for quality tests. In the case where 3 items are selected for performing quality tests, for example, quality tests a, b, and c may be selected, without limitation. In addition, random 2 elements can be selected from these 10 elements for quality tests. If 2 objects are selected for quality tests, for example, quality tests a and b,

quality tests a and c, quality tests a and d, quality tests a and e, quality tests a and f, quality tests a and g, quality tests a and h, quality tests a and i, quality tests a and j, quality tests b and c, quality tests b and d, quality tests b and e, quality tests b and f, quality tests b and g, quality tests b and h, quality tests b and i, quality tests b and j, quality tests c and d, quality tests c and e, quality tests c and f, quality tests c and g, quality tests c and h, quality tests c and i, quality tests c and j, quality tests d and e, quality tests d and f, d and g quality tests, d and h quality tests, d and i quality tests, d and j quality tests, e and f quality tests, e and g quality tests, e and h quality tests, e and i quality tests, quality tests e and j, quality tests f and i, quality tests f and j, or quality tests i and j, without limitation.

Producing culture

The next intermediate cell culture step is a culture step leading to the production of dental pulp cells enriched in pluripotent stem cells, which are the final products, and this step is specifically referred to as production culture.

The medium used for the production culture is preferably Dulbecco's modified Eagle's medium to which fetal bovine serum is added. The concentration (v/v%) of fetal bovine serum added to the medium is preferably 8% to 25% and more preferably 8% to 20%, and is, for example, 10%. An example of a detailed composition of Dulbecco's modified Eagle's medium (prior to the addition of fetal calf serum) is shown in Table 1. If desired, 3 to 5 mM, eg 4 mM, L-alanyl-L-glutamine can be added to the medium. In addition, an antibiotic is usually not added to the production culture. However, an antibiotic such as streptomycin can be added to the medium. In the case where streptomycin is added to the medium, streptomycin is added thereto so that the concentration of streptomycin in the medium is 10 mg/l to 250 mg/l, for example, 100 mg/l. In addition, each component can be replaced by its equivalent, such as its salt.

Productive cell culture may be performed in a cell culture plate, or may be performed on microcarriers in a cell culture device such as a bioreactor. In addition, the cells can be cultured and proliferated in a cell culture plate to further carry out microcarrier culture production in a cell culture device such as a bioreactor. In addition, in the case of culturing cells on microcarriers, a shake flask can be used instead of a bioreactor. In the case of culturing the cells in the cell culture plate in the production culture, the conditions are the same or similar to the culturing conditions of the above-described cells obtained by culturing the dental pulp suspension. That is, culturing the intermediate cells after thawing in the cell culture plate and harvesting the cells therefrom is repeated to increase the number of cells. Bioreactors commercially available from GE Healthcare, Merck KGaA, Sartorius AG and the like can be suitably used. Hereinafter, a cell culture method using a bioreactor as a cell culture apparatus will be described in detail.

In the case of culturing cells on microcarriers using a bioreactor in a production culture, the microcarriers to which the cells are attached are placed in the bioreactor, and the cells are cultured while stirring the microcarriers in the medium. Microcarriers are particles (small particles) of material to which cells can attach and are used to culture cells on their surfaces (including surfaces within pores in the case of porous microcarriers). In this sense, microcarriers may also be referred to as carrier particles or small carrier particles. The diameters (particle diameters) of the microcarriers during cell culture (i.e., during hydration) are preferably 50 to 400 µm and are, for example, 80 to 300 µm, 80 to 250 µm, 100 to 200 µm, 130 to 180 µm. The microcarriers may be porous, having pores directed inward from the surface of the particle. The diameters (pore diameters) of the porous microcarriers during cell culture are preferably 3 to 40 µm and are, for example, 5 to 25 µm, 10 to 20 µm, 5 to 15 µm.

The diameters (pore diameters) of the porous microcarriers during cell culture are preferably 3 to 40 µm and are, for example, 5 to 25 µm, 10 to 20 µm, 5 to 15 µm. Examples of microcarriers include microcarriers obtained by molding a polymeric compound into particles, ceramic particles, and glass particles. Examples of such a polymer compound include vinyl resin, urethane resin, epoxy resin, polystyrene, polymethyl methacrylate polyester, polyamides, polyimide, silicone resin, phenolic resin, melamine resin, urea resin, aniline resin, ionomer resin, polycarbonate, collagen, dextran, gelatin, cellulose , alginate and mixtures thereof. The surfaces of the microcarriers can be further modified with cell adhesion molecules and the like. Examples of such cell adhesion molecules include gelatin, keratin, elastin, heparan sulfate, dextran, dextran sulfate, chondroitin sulfate, sodium hyaluronate, n-isopropylacrylamide, collagen I to XIX, fibronectin, vitronectin, laminin-1 to -12, nidogen, tenascin, thrombospondin, osteopontin, fibrinogen, poly-D-lysine, poly-L-lysine, chitin, chitosan, sepharose, laminin, entactin I, or fragments or peptides containing cell adhesion domains of these cell adhesion molecules, and mixtures thereof. In addition, the specific gravity of the microcarriers in the medium is preferably close to 1, or from 0.9 to 1.2, and is, for example, from 0.9 to 1.1, or about 1.0.

Suitably, microcarriers can be used which are made from a gelatin matrix and have a particle diameter during cell culture of 130 to 180 µm and a pore diameter of 10 to 20 µm (or 5 to 15 µm).

Cell adhesion to microcarriers in the production culture is carried out by mixing cells with microcarriers while stirring them in solution. This stage is called the stage of cell adhesion to microcarriers. In the adhesion step, cells that come into contact with the microcarriers during agitation are successively attached to the surfaces of the microcarriers. The solution used in this step is preferably a cell culture medium, but the present invention is not specifically limited to this. Mixing of cells and microcarriers during the adhesion step can be performed intermittently. In this case, the cells and microcarriers, for example, are mixed for 1 minute to 20 minutes and then left for 10 minutes to 2 hours.

In case the adhesion step is performed using the cell culture medium as a solution, the step can be performed in a bioreactor. In the case of performing the adhesion step in the bioreactor, the cell culture medium, cells and microcarriers are placed and mixed in the bioreactor so that the cells adhere to the microcarriers. After cell adhesion, cell culture can be continued in the bioreactor.

Mixing of cells and microcarriers in the bioreactor at the stage of adhesion can be done intermittently. In this case, the cells and microcarriers are, for example, mixed for 1 minute to 20 minutes and then left for 10 minutes to 2 hours.

In general, a bioreactor refers to a biochemical reactor, such as a device for reacting with an immobilized enzyme, and a biological or microbiological reactor, such as a fermentation tank or a biological wastewater treatment device. The bioreactor used in the examples to be described later in the present invention is suitable for generating cells derived from dental pulp enriched in pluripotent stem cells to adhere to microcarriers and culture the cells with agitation in the medium. The bioreactor has a cell culture tank, medium stirrer, gas phase inlet, liquid phase inlet, drain port and sensor block. The culture tank is a tank having a substantially cylindrical cavity. In this state, it is possible to culture cells derived from dental pulp, enriched with pluripotent stem cells, to which microcarriers are attached in a culture tank. In the cavity of the culture tank, a stirrer is provided for the medium in the culture tank. The stirrer is, for example, an impeller, and the medium is stirred by rotating the impeller. The mixing of the medium with the help of an impeller is adjusted so that most of the microcarrier to which the cells are attached floats in the medium without settling on the lower surface of the cavity of the culture tank. The speed of rotation of the impeller must be appropriately selected depending on the shape of the culture vessel, the shape of the impeller, the types of microcarriers, etc., and is, for example, from 30 to 100 rpm, from 50 to 80 rpm, or about 70 rpm The gas phase inlet is designed to supply the gas phase such as air, oxygen and carbon dioxide to the culture tank. The gas phase inlet may have an outlet below the liquid level of the medium. In this case, the gas phase is released into the medium in the form of bubbles. The liquid phase inlet is designed to load various solutions, such as medium or glucose solution, into the culture tank. The drain port is designed to drain the medium into the culture tank. Media exits this drain port during media change. The drain port is also used to retrieve the microcarriers to which the cells are attached along with the medium. This drain port can be used when sampling microcarriers during culture or when collecting microcarriers after culture is complete. The sensor block includes sensors that measure the temperature, pH, and concentration of dissolved oxygen in the medium in the culture tank. The sensor unit may further include sensors that measure glucose and lactic acid concentrations. Sensors for measuring temperature, pH and concentration of dissolved oxygen in the solution, as well as the concentration of glucose and lactic acid, can be appropriately selected from those well known for installation.

Tank for cultivation of the bioreactor is fixed or installed in the bioreactor with the possibility of removal. Thanks to the removable culture tank, cleaning the culture tank is easy. Culture tanks are usually made of metal or hard resin and are cleaned and reused after use. Instead of such a culture tank, a flexible resin culture bag can be installed in the bioreactor, and the culture medium and cells can be placed in the bag. The culture bag is supported in the bioreactor, so it can be handled in the same way as a normal culture tank. Because the culture bag is disposable, no rinsing is required after use, which improves work efficiency during culture.

When the concentration of glucose in the medium in the production culture falls below a certain value, glucose is added to the medium. For example, in the case where the glucose concentration is less than or equal to 0.1 mM, glucose is added to the medium so that the final glucose concentration becomes 5 to 7 mM. In addition, if the concentration of lactic acids exceeds a certain value in the production culture, all or part of the medium is replaced. In the case where the concentration of lactic acids is, for example, greater than or equal to 20 mM, the entire medium is replaced or part of the medium is replaced so that the concentration of lactic acids becomes, for example, lower than or equal to 5 mM or less than or equal to 2 mM.

Oxygen, air and CO2 are fed into the medium through the gas phase inlet during cultivation in the bioreactor. The amount of oxygen supplied is controlled so that the concentration of dissolved oxygen in the environment is between 50% and 100% of the saturation concentration, for example, so that the concentration of dissolved oxygen is maintained at 50% or 100% of that. In addition, with regard to the flow rate of CO2, CO2 is supplied in such a way as to maintain the pH of the medium, for example, at a level of 7.0 to 7.8, so that the cells can grow appropriately. Dissolved oxygen concentration and pH are monitored over time. Therefore, when the controlled numerical values deviate from the set values, the concentration of dissolved oxygen and pH can be kept constant by reducing the amount of oxygen and CO2 supplied, respectively. In addition, as the cells proliferate, additional microcarriers can be fed into the bioreactor.

Cultivation in a bioreactor is carried out until the surfaces of the microcarriers are occupied by cells in a certain proportion. The share at this point can be appropriately taken as 20%, 40%, 85%, 95% or 98%, for example. In addition, cultivation in the bioreactor is carried out until the surfaces of the microcarriers are occupied by cells, until the density of cells on their surfaces becomes a certain value. The cell density at this point can be appropriately taken as 3,000 cells/cm ² , 5,000 cells/cm ² , 10,000 cells/cm ² , 20,000 cells/cm ² or 22,000 cells/cm ² , for example.

Cells to be frozen as a cell preparation derived from dental pulp preferably undergo at least 15 divisions after completion of the growth of the production culture, and more preferably undergo at least 20 divisions after harvesting as colony-forming cells in cell culture plate by culturing the dental pulp suspension. For example, cells that have undergone 15-30 divisions, 15-23 divisions, 15-20 divisions, 20-30 divisions, 20-28 divisions, or 23-26 divisions are cryopreserved.

Cells to be frozen as a cell preparation derived from dental pulp, after completion of growth of the production culture through intermediate cells, preferably undergo at least 10 divisions, and more preferably undergo at least 15 divisions after collection as cells, forming colonies in a cell culture plate by culturing the dental pulp suspension until the cells are considered intermediate cells. For example, cells that have undergone 13-20 divisions, 13-23 divisions, or 14-20 divisions are cryopreserved as intermediate cells. Then, in the production culture, which begins with the thawing of the intermediate cells, the cells preferably undergo at least 5 divisions until a pulp-derived cell preparation is obtained from the beginning of the production culture, and preferably undergo, for example , not less than 8 or not less than 10 divisions. In particular, when culture is carried out using microcarriers in the production culture, the cells preferably undergo 2 to 5 divisions, and preferably undergo 2 to 3 divisions, for example.

In the case where the cells are cultured in a cell culture plate, the average doubling time of the cells when the cells are cultured and undergo divisions until a dental pulp cell preparation is obtained from the intermediate cells is preferably within 96 hours, more preferably within 84 hours. , even more preferably within 72 hours, even more preferably within 48 hours, and even more preferably within 36 hours. In the case where the cells are cultured using microcarriers, the average doubling time of the cells when the cells are cultured and undergo divisions until the cell preparation obtained from the dental pulp is obtained from the intermediate cells is preferably within 8 days, more preferably within 6 days, even more preferably within 5 days and even more preferably within 4 days, and is, for example, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2.5 to 6 days or 2.5 to 5.5 days.

The standard can be set for the average doubling time of cells during the entire production period or during culture using microcarriers, and only cells having an average doubling time within the standard can be cryopreserved for use for later use. Such standards can be appropriately set, for example, within 84 hours, within 72 hours, within 48 hours, or within 36 hours in the case of culture in a cell culture plate, and within 8 days, within 6 days, in within 5 days, within 4 days, within 3 days, etc. in the case of cultivation using microcarriers. Cells that do not meet these standard values may be discarded without cryopreservation in order to selectively retain only cells that have the ability to undergo cell division a certain number of times or more as a cell preparation derived from dental pulp.

In production culture, cells proliferate until the number of pluripotent stem cells that can be obtained from one extracted tooth (for example, the third molar), until a cell preparation obtained from the pulp of the tooth, is preferably 1 × 10 ⁶ or more, more preferably 1 x 10 ⁷ or more, even more preferably 3 x 10 ⁷ or more, even more preferably 1 x 10 ⁸ or more, and even more preferably 1 x 10 ⁹ or more, and becomes, for example, from 1 x 10 ⁷ to 1 x 10 ¹⁰ . Theoretically, the number of cells undergoing 20 divisions, 25 divisions, and 30 divisions increases by 1 x 10 ⁶ times or more, 3 x 10 ⁷ times or more, and 1 x 10 ⁹ times or more.

Getting the drug

Microcarriers after completion of culture growth are collected and washed in a state in which cells are attached to them. The solution that can be used as a washing liquid for washing microcarriers is currently not particularly limited as long as it does not damage cells and does not inhibit the enzymatic activity of serine protease and metalloprotease, especially trypsin. However, for example, saline, PBS, low glucose DMEM (serum-free), and a wash solution having the composition shown in Table 4 may be used, and, for example, low glucose DMEM (serum-free) may be used. serum).

The washed microcarriers are treated with protease. The protease that can be used at this point is not particularly limited as long as it can decompose cell protein adhesion molecules, but serine protease, metalloprotease or a mixture thereof is preferred. Trypsin is one suitable serine protease and gelatinase is one suitable metalloprotease. The cells are released from the microcarriers by treatment with a protease. Furthermore, in the case where the materials of the microcarriers are proteins such as gelatin, the microcarriers themselves are degraded upon treatment with a protease. At this point, in the case where the microcarriers are porous, the cells that have adhered to the surfaces inside the pores are also released from the microcarriers. In the case of microcarriers made from a porous gelatin matrix, the gelatin is degraded by treatment with a protease and a suspension containing released cells and gelatin degradation products. In this case, trypsin, gelatinase or a mixture thereof is particularly suitable as a protease.

In the case where the microcarrier materials are proteins such as gelatin, a suspension is obtained containing cells released from the microcarriers due to protease and degradation products (gelatin degradation products in the case where the materials are gelatin) of proteins, which are microcarrier materials. . To prepare a preparation of isolated cells derived from dental pulp, it is preferable to remove protease and impurities, such as degradation products of microcarrier materials, from this suspension by a cell washing operation. In the following, the cell washing operation in the case where the microcarrier materials is gelatin will be described in detail.

The suspension resulting from the protease treatment can be washed by recollection by centrifugation and resuspension using the wash solution. Protease and milled gelatin degradation products can be removed by centrifugation. However, the degradation products of gelatin also include residues of a certain size, which are deposited along with the cells during centrifugation. Removal of such large gelatin residues can be carried out using membrane filtration. From this point of view, in the case of membrane filtration, the pore diameter of the filtration membrane is preferably 20 to 80 µm. For example, a filter membrane with a pore diameter of 25 µm, 26 µm, 27 µm, 28 µm, 30 µm, 50 µm, 70 µm, or 80 µm can be used. Filtration with a filter membrane can be effectively performed by preventing clogging of filter membranes by filtering with a filter membrane having a large pore diameter and then filtering with a filter membrane having a small pore diameter. For example, filtration may be first performed with a filter membrane having a pore diameter of 60 to 100 μm and then performed with a filter membrane having a pore diameter of 25 to 50 μm, or may be first performed with a filter membrane having a pore diameter of 60 to 75 µm, and then made with a filter membrane having a pore diameter of 25 to 30 µm. Filtration with a filter membrane can also be carried out by filtering only with a filter membrane with a small pore diameter. The pore diameter of the filter membrane used in this case is preferably 25 to 50 µm, and more preferably 25 to 30 µm. For example, a filter membrane with a pore diameter of 25 µm, 26 µm, 27 µm, 28 µm, or 30 µm is used. At this point, large gelatin residues do not pass through the filter membrane, and cells collect in the filtrate. However, the solution in which the cells are suspended can be replaced with a cryopreservation liquid (cryopreservation liquid for cell preparation) to be described below using an ultrafiltration membrane or the like without collecting the cells to prepare a pre-frozen preparation cell suspension.

The washing solution used in the above washing operation is designed to sufficiently remove the medium and protease. In the case where the microcarriers are proteins such as gelatin, a wash solution is also used to remove its degradation products. The composition of such a wash solution is not particularly limited. Saline saline, phosphate buffered saline, Ringer's acetate solution, Ringer's bicarbonate solution, and the like can be used as the wash solution, but it is preferable to use a solution prepared by adding human serum albumin to Ringer's bicarbonate solution. Commercially available acetate Ringer's solutions and bicarbonate Ringer's solutions can be used. For example, PLASMA-LYTE A (Baxter) and Physio 140 (Otsuka Holdings Co., Ltd.) can be used as Ringer's acetate solutions, and BICARBON Injection (AJINOMOTO CO., INC.) can be used as Ringer's bicarbonate solution. Suitable examples of component composition and electrolyte concentration of Ringer's bicarbonate solution are shown in Tables 2 and 3, respectively.

A suitable example of the component composition of the wash solution is shown in Table 4. In addition, Table 5 shows a suitable example of the concentration of the electrolyte contained in the wash solution. Sodium acetyltryptophan and sodium caprylate can also be excluded from the components of the wash solution presented in tables 4 and 5.

In the case of performing centrifugation and membrane filtration to remove impurities after protease treatment, centrifugation may be performed first, followed by membrane filtration, or membrane filtration may be performed, followed by centrifugation. However, it is preferable to carry out membrane filtration first and then centrifugation.

The harvested cells, after washing with the wash solution, are suspended in a cryopreservation liquid (cryopreservation liquid for cell preparation). A dental pulp-derived cell preparation obtained by encapsulating and freezing this suspension (cell suspension for pre-freezing) in a cell cryopreservation container is the optimal form for storage or transportation among dental pulp-derived cell preparations. The composition of the solution part (cryopreservation liquid for cell preparation) of the cell suspension for pre-freezing is not particularly limited, provided that mammalian cells, in particular cells derived from dental pulp enriched in human pluripotent stem cells, can be frozen and thawed without death. The cell cryopreservation fluid for cell preparation contains a cytoprotective agent. Cytoprotective agents have, for example, the function of inhibiting the formation of ice crystals inside cells and the destruction of cells when cells are frozen. Suitable examples of cytoprotective agents include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin and glycerol. Two or more of these can be combined and used as a cytoprotective agent. In the case of using dimethyl sulfoxide as a cytoprotective agent, its concentration is preferably from 5% to 15% (v/v), and more preferably from 9% to 11% (v/v), and is, for example, 10% (vol./vol.). The cryopreservation fluid may include a buffering agent. A buffering agent that can adjust the pH of the aqueous solution from 6 to 8, for example 6.8 to 7.8, is preferred. Examples of such buffering agents include those containing carbonate ions, bicarbonate ions, citrate ions, and sodium ions. The cryopreservation fluid for cell preparation may further comprise human serum albumin. In the case of using human serum albumin, its concentration is preferably 40 to 100 g/l, and more preferably 46 to 56 g/l, and may be, for example, 51 g/l.

The product obtained by adding a solution of human serum albumin and dimethyl sulfoxide to Ringer's bicarbonate solution is a suitable example of a cryopreservation fluid for cell preparation. Cryopreservation liquid for cell preparation contains 59 to 80.4 mM sodium chloride, 2.3 to 3.08 mM potassium chloride, 0.85 to 1.16 mM calcium chloride dihydrate, 0.28 to 0.385 mM hexahydrate magnesium chloride, 14 to 19.2 mM sodium bicarbonate, 0.94 to 1.28 mM sodium. citrate dihydrate, 46 to 56 g/l human serum albumin, 3.73 to 4.55 mM sodium acetyltryptophan, 3.74 to 4.58 mM sodium caprylate, and 9% to 11% (v/v ) DMSO. The intermediate cell cryopreservation liquid shown in Table 6 can also be suitably used as the cryopreservation liquid for cell preparation.

Thus, a cryopreservation liquid for preparing cells can be suitably used, which can be essentially used as a cryopreservation liquid for intermediate cells. For example, its composition is: 65.8 to 80.4 mM sodium chloride, 2.52 to 3.08 mM potassium chloride, 0.95 to 1.16 mM calcium chloride dihydrate, 0.315 to 0.385 mM hexahydrate magnesium chloride, 15.7 to 19.2 mM sodium bicarbonate, 1.04 to 1.28 mM sodium citrate dihydrate, 46 to 56 g/l human serum albumin, 3.73 to 4.55 mM sodium acetyltryptophan , 3.74 to 4.58 mM sodium caprylate and 9 to 11% (v/v) DMSO. In addition to the composition shown in Table 6, another composition is, for example, 70.8 to 71.3 mM sodium chloride, 2.71 to 2.73 mM potassium chloride, 1.01 to 1.02 mM calcium chloride dihydrate, 0.335 to 0.345 mM magnesium chloride hexahydrate, 16.9 to 17.0 mM sodium bicarbonate, 1.12 to 1.14 mM sodium citrate dihydrate, 53.6 to 54.3 g/l whey human albumin, 4.36 to 4.41 mM sodium acetyltryptophan, 4.37 to 4.43 mM sodium caprylate, and 10.6 to 10.9% (v/v) DMSO. Among them, sodium acetyltryptophan and sodium caprylate can be excluded. The ratio of the osmotic pressure of the cryopreservative liquid for cell preparation to saline is preferably 0.9 to 1.1.

In addition, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin, and dimethyl sulfoxide can be suitably used as intermediate cell cryopreservation liquid. For example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, in concentrations from 91 to 113 mm, from 2.52 to 3.08 mm, from 0.95 to 1.16 mM, 0.315 to 0.385 mM, 15.6 to 19.2 mM, 1.04 to 1.28 mM, 46 to 56 g/L and 9% to 11% ( v/v) is a suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, in concentrations from 100 to 102 mm, from 2.71 to 2.77 mM, 1.01 to 1.03 mM, 0.335 to 0.345 mM, 16.9 to 17.2 mM, 1.13 to 1.15 mM, 52.2 to 54.3 g/L and from 10.3 to 10.9% (v/v) is another suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, at concentrations of 102 mm, 2.80 mm, 1.05 mm, 0 .35 mM, 17.4 mM, 1.16 mM, 51 g/l and 10% (v/v) is another suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, at concentrations of 101 mm, 2.77 mm, 1.03 mm, 0 .34 mM, 17.2 mM, 1.15 mM, 52 g/l and 10% (v/v) is another suitable example of this. In addition, for example, a solution containing sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, citrate ions, human serum albumin and dimethyl sulfoxide, respectively, at concentrations of 100 mm, 2.71 mm, 1.01 mm, 0.34 mM, 16.9 mM, 1.13 mM, 53.6 g/l and 10.7% (v/v) is another suitable example of this.

In the case where the cryopreservation liquid for preparing cells further contains acetyltryptophan or a salt thereof, its concentration is preferably 3.73 to 4.55 mM, and more preferably 4.24 to 4.41 mM, and is, for example, 4, 14 mM, 4.24 mM or 4.36 mM. In the case where the cell preparation cryopreservation liquid further contains caprylic acid or a salt thereof, its concentration is preferably 3.74 to 4.58 mM, and more preferably 4.25 to 4.43 mM, and is, for example, 4. 16 mM, 4.25 mM or 4.37 mM.

The cell density of the suspension in which the pluripotent stem cell-enriched dental pulp-derived cells are suspended in the cell preparation cryopreservation liquid is not particularly limited, but is preferably 5 x 10 ⁶ to 8 x 10 ⁷ cells/mL more preferably from 1.5 x 10 ⁷ to 3 x 10 ⁷ cells/ml and even more preferably from 2.1 x 10 ⁷ to 3.0 x 10 ⁷ cells/ml, and is, for example, 2.5 x 10 ⁷ cells/ ml. Suspended pluripotent stem cells derived from dental pulp are placed in a cell cryopreservation container and then subjected to cryopreservation.

The composition of the cell suspension in which cells derived from dental pulp enriched in pluripotent stem cells are suspended in cryopreservation liquid for cell preparation varies depending on the density of the suspended cells, but mainly includes 59-61 mM sodium chloride, from 2.3 to 2.6 mM potassium chloride, 0.85 to 0.98 mM calcium chloride dihydrate, 0.28 to 0.32 mM magnesium chloride hexahydrate, 14 to 16 mM sodium bicarbonate, 0.94 to 1.08 mM sodium citrate dihydrate, 49 to 50 g/l human serum albumin, 4.0 to 4.1 mM sodium acetyltryptophan, 4.0 to 4.1 mM sodium caprylate, and 9% to 11% (v/v .) DMSO.

The amount of cell suspension to be dispensed into one cell cryopreservation container needs to be adjusted accordingly, and is preferably 1 to 20 ml. Further, the number of cells to be placed in one cell cryopreservation container is preferably 5 x 10 ⁶ to 9.2 x 10 ⁸ .

Suitable containers for cryopreserving cells suspended in a cryopreserving liquid for cell preparation, as well as procedures and conditions for freezing and preserving cells, are the same as those already described for cryopreserving intermediate cells.

The cells cryopreserved in this way can be used as pharmaceuticals (dental pulp-derived cell preparations) containing, as active components, dental pulp-derived cells enriched in pluripotent stem cells. In this case, dental pulp-derived cells enriched in pluripotent stem cells are transported in a frozen state and thawed when used for administration to a patient.

The viability of the cells contained in the cell preparations from the dental pulp obtained by the production culture immediately after thawing is preferably greater than or equal to 50%, more preferably greater than or equal to 60%, and even more preferably greater. than or equal to 70%, and preferably, for example, greater than or equal to 80%, greater than or equal to 90%, or greater than or equal to 95%. As dental pulp-derived cell preparations, which are pharmaceutical preparations, only cells having a certain viability value or a higher viability value can be used in accordance with properly established standards.

When cells contained in dental pulp-derived cell preparations are cultured in a medium containing a substance known to induce differentiation into chondrocytes, an increase in the expression level of aggrecan as a whole is observed. In addition, when cells are cultured in a medium containing a substance known to induce differentiation into osteocytes, an increase in the amount of calcium accumulated in the cells is generally observed. That is, cells have the ability to differentiate into chondrocytes and osteocytes when observed as a whole.

In one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one of CD73, CD90, CD105, and CD166 in surface antigen marker expression patterns. Similarly, the cells are negative for at least one of CD34 and CD45. For example, when the cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for CD73 and CD90 and negative for CD34 or, for example, positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in patterns of expression of surface antigen markers immediately after cell thawing or during cell cultivation after thawing.

In addition, in one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are negative for at least one of CD40, CD80, CD86 and MHC class II antigen in expression patterns. surface antigen markers. For example, when cells contained in dental pulp-derived cell preparations are observed as a whole, cells are negative for CD40, CD80, CD86, and MHC class II antigen in surface antigen marker expression patterns immediately after the cells are thawed or when the cells are cultured. after defrosting. It is known that when cells positive for these surface antigen markers are transplanted into allogeneic individuals, the cells tend to be recognized as an antigen to be eliminated from the living body. In the case where the cells are negative for at least one of these surface antigen markers, this means that the pulp-derived cells enriched in pluripotent stem cells are hypoimmunogenic to that extent and tend and are unlikely to be excluded from living organism when transplanted into allogeneic individuals. In addition, cells can remain negative for CD40, CD80 and CD86 and be positive for the MHC class II antigen in surface antigen marker expression patterns when the cells are stimulated with IFN-γ. Cells remain, for example, negative for CD40, CD80, and CD86, and become positive for MHC class II antigen when, for example, the cells are stimulated with IFN-γ.

In addition, in one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are, for example, positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC-class II antigen in expression patterns of surface antigen markers immediately after cell thawing or during cell culture after thawing. Cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ.

In one embodiment, regarding characteristic (d-1), when cells contained in cell preparations derived from dental pulp are observed as a whole, the cells are positive for at least one, preferably for all of CD29, CD44, CD59 and CD164 in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (d-2), when the cells contained in the cell preparations obtained from the dental pulp are observed as a whole, the cells are positive for at least one, preferably for all of CD9, CD13, CD46, CD47, CD58, CD63, CD73, CD81, CD90, CD98, CD147, HLA-A, -B and -C and EGF-R in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (d-3), when the cells contained in the cell preparations obtained from the dental pulp are observed as a whole, the cells are positive for at least one, preferably for all of the CD49b, CD49c, CD49e, CD55, CD95, CD151 and CD166 in surface antigen marker expression patterns.

Herein, when individual cells are observed included in all cells, preferably at least 65%, more preferably at least 75%, and even more preferably at least 80% of the observed cells are positive for these antigens .

In addition, in one embodiment, regarding characteristic (d-4), when the cells contained in the cell preparations obtained from the dental pulp are observed as a whole, the cells are positive for at least one, preferably for all of the CD10, CD49f, CD105 and CD140b in expression patterns of surface antigen markers.

Herein, when individual cells are observed included in all cells, preferably at least 60%, more preferably at least 70%, and even more preferably at least 75% of the observed cells are positive for these antigens .

In one embodiment, the cells contained in the dental pulp-derived cell preparations preferably have two or more, more preferably three or more, and even more preferably all of the characteristics presented in (d-1), (d-2) above, (d-3) and (d-4). For example, cells having the characteristics described in (d-1) and (d-2) above are a suitable embodiment of the present invention.

In one embodiment, regarding characteristic (d-5), when the cells contained in the cell preparations obtained from the dental pulp are observed as a whole, the cells are negative for at least one, preferably for all of CD1a, CD1d, CD2 , CD3, CD4, CD5, CD7, CD8a, CD8b, CD11b, CD11c, CD15, CD15s, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD28, CD30, CD31, CD32, CD33, CD34 , CD35, CD37, CD38, CD41a, CD86, CD87, CD88, CD89, CDw93, CD94, CD100, CD102, CD103, CD114, CD117, CD118, CD120b, CD121b, CD122, CD123, CD124, CD126, CD127, CD128b, CD132 , CD134, CD135, CD137, CD137, CD138, CD144, CD150, CD153, CD154, CD158a, CD158b, CD161, CD162, CD163, CD172b, CD177, CD178, CD180, CD184, CD195, CD205, CD19 CD210, CD 212, CD220 , CD226, CD229, CD231, CD235a, CD244, CD255, CD267, CD268, CD278, CD279, CD282, CD294, CD305, CD309, CD314, CD321, CDw327, CDw328, CD329, CD335, CD335, CD335, BLTR-1, CLIP , CMRF-44, CMRF-56, fMLP-R, SSEA-1, TRA-1-60, CLA, β7 integrin, and invariant NKT in expression patterns of surface antigen markers.

Here, when single cells are observed, included in all cells, preferably at least 10%, more preferably at least 5%, and even more preferably at least 2%, and even more preferably at least 1% of the observed cells are positive for these antigens. .

In one embodiment, regarding characteristic (d-6), when cells contained in cell preparations derived from dental pulp are observed as a whole, the cells are negative for at least one, preferably for all of CD1b, CD6, CD27 , CD41b, CD42a, CD42b, CD43, CD45, CD45RB, CD48, CD50, CD53, CD57, CD62E, CD62L, CD62P, CD64, CD66b, CD66f, CD69, CD70, CD72, CD75, CD84, CD85, CD97, CD99R, CD183 , CD193, SSEA-3 and γδ TCR in expression profiles of surface antigen markers.

Here, when single cells included in all cells are observed, preferably only at most 10%, more preferably only at least 5%, and even more preferably only at least 2% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (d-7), when cells contained in cell preparations derived from dental pulp are observed as a whole, the cells are negative for at least one, preferably for all of CD4v4, CD14, CD36 , CD45RA, CD45RO, CD66 (a, c, d, e), CD79b, CD83, CD106, CD152, CD209, CD271, CD275, CD326, MIC A/B and αβ TCR in surface antigen marker expression profiles.

Here, when single cells are observed, included in all cells, preferably at least 10% and more preferably at least 5% of the observed cells are positive for these antigens.

In one embodiment, regarding characteristic (d-8), when cells contained in cell preparations derived from dental pulp are observed as a whole, the cells are negative for at least one, preferably for all of CD26, CD40, CD56 , CD80, CD146 and MHC-class II antigen in expression profiles of surface antigen markers.

Here, when single cells included in all cells are observed, preferably only at most 20% and more preferably only at least 10% of the observed cells are positive for these antigens.

In one embodiment, the cells contained in the dental pulp-derived cell preparations preferably have two or more, more preferably three or more, and even more preferably all of the characteristics presented in (d-1), (d-2), ( d-3), (d-4), (d-5), (d-6), (d-7) and (d-8). For example, cells having the characteristics shown in (d-1) and (d-5) above are a suitable embodiment of the present invention.

In addition, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF) immediately after thawing or during culture after thawing, and the expression level prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells.

In addition, in one embodiment, when cells contained in cell preparations derived from dental pulp are cultured in the presence of IFN-γ, the amount of kynurenine secreted increases.

In one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one or all of CD47, CD81, and CD147 and negative for at least one or all of CD19, CD34 and CD206 in surface antigen marker expression profiles. In addition, in one embodiment of the present invention, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one or all of CD47, CD81, and CD147 and negative for at least at least one or all of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a and SSEA-1 in surface antigen marker expression profiles.

In one embodiment, the cells contained in the dental pulp-derived cell preparations are negative for at least one, eg, all of CD19, CD26, CD34, CD56, CD106, CD117, CD146, and CD271.

In one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, these cells are positive for at least one, e.g., all of CD140b and HLA-A, -B and -C in expression profiles of surface antigen markers.

In one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one, e.g., all of CD10, CD49e, and CD95 in surface antigen marker expression profiles.

In one embodiment of the present invention, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one, eg, all of CD46, CD47, CD55, CD58. and CD59 in surface antigen marker expression profiles.

In one embodiment of the present invention, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are, for example, positive for at least one of CD73, CD90, CD105, and CD166, and negative for at least one of CD34 and CD45 in surface antigen marker expression profiles.

In addition, when cells contained in dental pulp-derived cell preparations are observed in general immediately after thawing or in culture after thawing, the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen in expression profiles of surface antigen markers. In addition, the cells remain negative for CD40, CD80 and CD86 and become positive for the MHC class II antigen when the cells are stimulated with IFN-γ. In addition, cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and secreted prostaglandin E2 (PGE2) is increased by stimulation of TNF-α cells.

When cells contained in dental pulp-derived cell preparations are cultured, the cells secrete various humoral factors.

In one embodiment, regarding characteristic (d-9), when cells contained in cell preparations derived from dental pulp are cultured, the cells express at least one, preferably all, of MMP-2, IGFBP-4, and cystatin. WITH.

In addition, regarding characteristic (d-10), in one embodiment, when cells contained in cell preparations derived from dental pulp are cultured, the cells express at least one, preferably all of IL-6, IL- 11, MCP-1, IL-8, GROα, HGF, VEGF, VCAM-1, TIMP-3, TIMP-2 and TIMP-1.

In addition, regarding characteristic (d-11), in one embodiment, when cells contained in cell preparations derived from dental pulp are cultured, the cells express at least one, preferably all of IL-23, IFN- a, TNF-α, IL-18, IL-27, TARC, ENA-78, MIP-3α, MIP-1β, IP-10, SCF and ICAM-1.

In addition, regarding characteristic (d-12), in one embodiment, when cells contained in cell preparations derived from dental pulp are cultured, the cells do not express or almost do not express at least one, preferably all of IL - 21, eotaxin, MIP-1a, MIG, I-TAC, IP-10 and GM-CSF.

In addition, regarding the characteristic (d-13), in one embodiment, when cells contained in cell preparations obtained from dental pulp are cultured, then at least one of the expression levels of IL-6, IL-23, IL-11 , MCP-1, ENA-78, HGF, VEGF, MMP-2, IGFBP-4, cystatin C, TIMP-3, TIMP-2 and TIMP-1 are increased compared to the level of expression in intermediate cells. For example, the level of expression of IL-6, IL-11, HGF, TIMP-3 and TIMP-1 is increased compared to the level of expression in intermediate cells.

In addition, regarding characteristic (d-14), in one embodiment, when the cells contained in cell preparations obtained from dental pulp are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-6 is higher, than in the case of culturing cells in the absence of such.

In addition, regarding characteristic (d-15), in one embodiment, in the case where cells contained in cell preparations obtained from dental pulp are cultured in the presence of TNF-α or IFN-γ, the expression level of IL-11 is higher, than in the case of cultivation of cells in the absence of such.

In addition, regarding characteristic (d-16), in one embodiment, when cells contained in cell preparations obtained from dental pulp are cultured in the presence of TNF-α or IFN-γ, the expression level of IP-10 is higher than in the case of culturing cells in the absence of such.

In addition, regarding characteristic (d-17), in one embodiment, when the cells contained in cell preparations obtained from dental pulp are cultured in the presence of TNF-α or IFN-γ, the expression level of MCP-1 is higher, than in the case of culturing cells in the absence of such.

Further, with respect to characteristic (d-18), in one embodiment, GM-CSF expression is induced when cells contained in dental pulp-derived cell preparations are cultured in the presence of TNF-α, but GM-CSF expression is not induced when cells cultivated in the presence of IFN-γ.

In addition, regarding characteristic (d-19), in one embodiment, the expression level of HGF is reduced when cells contained in cell preparations derived from dental pulp are cultured in the presence of TNF-α compared to the case when the cells are cultured in its absence, while the expression level of HGF is increased when cells are cultured in the presence of IFN-γ compared to when cells are cultured in its absence.

In addition, regarding characteristic (d-20), in one embodiment, when the cells contained in the cell preparations obtained from dental pulp are cultured in the presence of TNF-α, the expression level of IL-8 is higher than when cells are cultured in their absence.

In one embodiment of the invention, the pluripotent stem cell-enriched pulp-derived cells of the present invention preferably have two or more, more preferably three or more, even more preferably four or more, and even more preferably all of the characteristics described above ( d-9) - (d-20). For example, cells having the characteristics described in (d-9) and (d-16) above and cells having the characteristics presented in (d-9), (d-15) and (d-16) described above , represent a suitable embodiment of the present invention.

In addition, when cells contained in dental pulp-derived cell preparations are observed as a whole immediately after thawing or in culture after thawing, the expression level of aggrecan is increased when the cells are cultured in a medium containing a substance known to induces differentiation into chondrocytes. In addition, when cells derived from dental pulp enriched in pluripotent stem cells are cultured in a medium containing a substance known to induce differentiation into osteocytes and observed as a whole, the amount of calcium accumulated in the cells increases. That is, cells derived from the dental pulp, enriched with pluripotent stem cells, have the ability to differentiate into chondrocytes and osteocytes. The fact that cells contained in cell preparations derived from dental pulp have the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes can be investigated, respectively, using the methods described in Examples 20 and 19. Only groups of cells in which the ability to differentiate into chondrocytes and the ability to differentiate into osteocytes using these methods can be supplied to medical institutions as products.

According to suitably established standards, only cells showing predetermined surface antigen marker expression patterns and/or gene expression patterns may be stored as dental pulp-derived cell preparations.

In addition, when cells contained in dental pulp-derived cell preparations are observed in general immediately after thawing or in culture after thawing, these cells are mainly referred to as dental pulp-derived pluripotent stem cells. Dental pulp-derived pluripotent stem cells in attached culture are observed under an optical microscope as essentially spindle-shaped attached cells. The proportion of essentially fusiform cells in all cells of the attached culture, as viewed under an optical microscope, is preferably greater than or equal to 99%, more preferably greater than or equal to 99.5%, even more preferably greater than or equal to 99.9%. and even more preferably greater than or equal to 99.95%. Only dental pulp-derived cell preparations having a certain value or a high proportion of essentially spindle cells can be supplied to medical facilities as a product in accordance with properly established standards.

In addition, when cells contained in dental pulp-derived cell preparations are cultured after thawing, the cells have the ability to undergo at least 3 cell divisions, preferably have the ability to undergo at least 4 cell divisions, more preferably undergo at least 5 cell divisions, and even more preferably the ability to undergo at least 10 cell divisions. In addition, the average doubling time of dental pulp-derived cell preparations when thawed and cultured in a cell culture plate is preferably in the range of 96 hours, more preferably in the range of 84 hours, even more preferably in the range of 72 hours, even more preferably in range of 48 hours, and even more preferably in the range of 36 hours. At this point, cell culture conditions are used that are the same or equivalent to those of the above-described pluripotent stem cell-enriched dental pulp-derived cells described, for example, in Example 5. Only dental pulp-derived cell preparations having the ability to undergo cell divisions a certain number of times or more, can be supplied to medical facilities as a product in accordance with properly established standards. In addition, only dental pulp-derived cell preparations having an average doubling time within a certain time can also be supplied to medical institutions as a product in accordance with properly established standards. Such standards are, for example, cells having the ability to undergo at least 3 cell divisions and an average doubling time of 72 hours, cells having the ability to undergo at least 4 cell divisions and an average doubling time of 72 hours, and cells having the ability to undergo at least 5 cell divisions and an average doubling time of 72 hours.

That is, the cells contained in the dental pulp-derived cell preparations retain the properties of stem cells, which are highly dividing and can differentiate into two or more cell types. Intermediate cells to be used in the next culture step may also be referred to as cells having a high colony-forming capacity.

Cells contained in cell preparations derived from dental pulp preferably have undergone at least 16 cell divisions in an in vitro medium. For example, cells have undergone at least 21, 22, or 23 cell divisions.

When the cells contained in the dental pulp-derived cell preparations are thawed, the average cell diameter in the state in which the cells are in suspension culture in solution, for example, in a medium, is less than or equal to 20 μm or less than or equal to 18 μm, and is , for example, 10 to 20 µm, 10 to 18 µm, 12 to 20 µm, 14 to 20 µm, and 16 to 20 µm.

Examples of qualitative tests of cells contained in cell preparations derived from dental pulp are presented below as qualitative tests a' - k'. Qualitative tests are conducted on one or more items. All of these items can be completed. The cells that will be used for qualitative tests at present are either cells (1) as a cell preparation obtained from the dental pulp, obtained by fractionating some cells before they are suspended in a cryopreservation liquid, cells (2) as a cell preparation , obtained from dental pulp, obtained by fractionating some cells before suspending them in a cryopreservation liquid and then subculturing these fractionated cells, cells (3), as a cell preparation obtained from dental pulp, before freezing, which were suspended in a cryopreservation liquid for freezing, cells (4) as a cell preparation obtained from dental pulp immediately after thawing of frozen cells, or cells (5) as a cell preparation obtained from dental pulp, obtained by thawing frozen cells and further culturing thawed cells. Unless otherwise noted, the same qualitative test may be performed on two or more of these cells (1) - (5).

(Qualitative test a') Checking that the proportion of the number of cells showing a substantially fusiform shape in all cells when observed under an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9 %, or greater than or equal to 99.95.

(Qualitative test b') Checking that cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90% or greater than or equal to 95%.

(Qualitative test c') Checking that cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression profiles, or checking that cells are positive for at least one of CD73 and CD90 and are negative for CD34 in expression profiles of cell surface antigen markers.

(Qualitative test d') Checking that cells are negative for CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression profiles, or checking that cells are negative for at least CD40, CD80, CD86 and MHC-class II antigen in expression profiles of cell surface antigen markers.

(Qualitative test e') Checking that cells remain negative for CD40, CD80 and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression profiles when cells are stimulated with IFN-γ, or checking at least one of that cells are CD40 negative, CD80 negative, CD86 negative, and MHC class II antigen negative in cell surface antigen marker expression profiles when cells are stimulated with IFN-γ.

(Qualitative test f) Checking that cells express prostaglandin E2 (PGE2) and/or vascular endothelial growth factor (VEGF), and the expression level of prostaglandin E2 (PGE2) is increased by stimulating TNF-α cells.

(Qualitative test g') Checking that the expression level of aggrecan is increased when cells are cultured in a medium containing a substance known to induce differentiation into chondrocytes.

(Qualitative test h') Checking that the amount of calcium accumulated in cells is increased when cells are cultured in a medium containing a substance known to induce differentiation into osteocytes.

(Qualitative test i') Checking that the cells have the ability to undergo at least 3, 4 or 5 cell divisions in the cell culture plate.

(Qualitative test j) Checking that the average cell doubling time in the cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours or 36 hours during the qualitative test i.

(Qualitative test k') Checking that the average cell diameter in the state when the cells are in suspension culture is less than or equal to 20 μm, less than or equal to 18 μm, 10 to 20 μm, 10 to 18 μm, 12 to 20 µm, 14 to 20 µm or 16 to 20 µm.

The above qualitative tests can be performed on arbitrary 10 items (Qualitative test a') - (Qualitative test j'). In addition, random 9 items can be selected from these 10 items to conduct qualitative tests. In case 9 items are selected for qualitative tests, for example, qualitative tests a', b', c', d', e', f, g', i' and j' can be selected, without any restrictions . In addition, random 8 elements can be selected from these 10 elements to conduct qualitative tests. In the case where 8 items are selected for qualitative tests, for example, qualitative tests a', b', c', d', e', f, i' and j' can be selected without any restrictions. In addition, random 7 items can be selected from these 10 items to conduct qualitative tests. In the case where 7 items are selected for qualitative tests, for example, qualitative tests a', b', c', d', e', i' and j' can be selected without any restrictions. In addition, random 6 items can be selected from these 10 items to conduct qualitative tests. In the case where 6 items are selected to carry out qualitative tests, one can select, for example, qualitative tests a’, b’, c’, d’, i’ and j’, without any restrictions. In addition, random 5 items can be selected from these 10 items to conduct qualitative tests. In case 5 items are selected for conducting qualitative tests, for example, qualitative tests a’, b’, c’, i’ and j’ can be selected without any restrictions. In addition, random 4 items can be selected from these 10 items to conduct qualitative tests. In the case where 4 items are selected for qualitative tests, for example, qualitative tests a', b', c' and j' can be selected without any restrictions. In addition, random 3 items can be selected from these 10 items to conduct qualitative tests. In the case where 3 items are selected to conduct qualitative tests, for example, qualitative tests a, b and c can be selected without any restrictions. In addition, random 2 items can be selected from these 10 items to conduct qualitative tests. In the event that 2 items are selected to conduct qualitative tests, for example, qualitative tests a ' and b', qualitative tests a ' and c', qualitative tests a ' and d', qualitative tests a ' and e', qualitative tests a' and f', qualitative tests a' and g', qualitative tests a' and h', qualitative tests a' and i', qualitative tests a' and j', qualitative tests b' and c ', quality tests b' and d', quality tests b' and e', quality tests b' and f', quality tests b' and g', quality tests b' and h', quality tests b and i, quality tests b and j, qualitative tests c and d, qualitative tests c and e, qualitative tests c' and f, qualitative tests c' and g', qualitative tests c' and h', qualitative tests c' and i', qualitative tests c ' and j ', qualitative tests d 'and e', qualitative tests d' and f, qualitative tests d' and g', qualitative tests d' and h', qualitative tests d' and i', qualitative tests d' and j ', qualitative tests e' and f, qualitative tests e' and g', qualitative tests e' and h', qualitative tests e' and i', qualitative tests e' and j', qualitative tests f and i', qualitative tests f and j ' , or qualitative tests i' and j'.

In a method for obtaining cells derived from dental pulp enriched in pluripotent stem cells of the present invention, pluripotent stem cells contained in dental pulp are cultured, proliferated and cryopreserved as intermediate cells, and then thawed, further cultured and proliferated to form a cell preparation, obtained from the dental pulp. The process to obtain a dental pulp-derived cell preparation from intermediate cells includes the step of culturing the pluripotent stem cells in a state in which the pluripotent stem cells are attached to the surface of the particles. Due to the inclusion of such a step, in one embodiment, the cells contained in the cell preparations derived from dental pulp have different properties from the intermediate cells.

For example, with regard to the positivity ratios of surface antigens, when cells contained in cell preparations derived from dental pulp are observed as a whole, the cells have high positivity ratios for any one or all of CD39, CD49a, CD61, CD107a, CD107b and CD143 in expression profiles of surface antigen markers compared to intermediate cells. For example, cells contained in dental pulp-derived cell preparations have a high CD107b positivity rate compared to intermediate cells. The positivity ratio of these cell surface antigens contained in dental pulp-derived cell preparations is preferably at least 10% higher and more preferably at least 20% higher than intermediate cells. In addition, cells contained in dental pulp-derived cell preparations have a low CD146 positivity rate compared to intermediate cells. The cell positivity ratio for CD146 in cells contained in cell preparations derived from dental pulp is at least 20% less or at least 50% less than intermediate cells.

In addition, for example, with regard to expression levels of humoral factors, cells contained in cell preparations derived from dental pulp have high levels of expression of any one or all of IL-6, IL-23, IL-11, MCP-1, ENA-78, HGF, VEGF, MMP-2, IGFBP-4, cystatin C, TIMP-3, TIMP-2 and TIMP-1 compared to intermediate cells. In particular, cells contained in dental pulp-derived cell preparations have high levels of expression of one or all of IL-6, IL-11, HGF, IGFBP-4, TIMP-3, and TIMP-1 compared to intermediate cells. . In particular, cells contained in dental pulp-derived cell preparations have high levels of expression of one or all of IL-6 and HGF compared to intermediate cells.

Intermediate cells and cells contained in dental pulp-derived cell preparations retain the properties of stem cells, which are highly dividing and can differentiate into two or more cell types. These cells can also be referred to as cells having a high colony-forming capacity.

When intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are, for example, positive for at least one of CD73, CD90, CD105, and CD166 and negative for at least one of CD34 and CD45 in the expression profiles of surface antigen markers. For example, intermediate cells and cells contained in cell preparations derived from dental pulp are, for example, positive for CD73 and CD90 and negative for CD34 in surface antigen marker expression profiles. These expression profiles are common to mesenchymal stem cells. However, these expression profiles also have characteristic properties.

In one embodiment of the present invention, when intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one or all of CD47, CD81 and CD147 and negative for at least at least one or all of CD19, CD34 and CD206 in the expression profiles of surface antigen markers. In addition, in one embodiment of the present invention, when pluripotent stem cell-enriched dental pulp-derived cells are observed in general, these cells are positive for at least one or all of CD47, CD81, and CD147 and negative, at least one or all of CD19, CD31, CD33, CD34, CD38, CD45, CD206, CD235a and SSEA-1 in surface antigen marker expression profiles.

In one embodiment, when cells contained in intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are negative for at least one of, e.g., all of CD19, CD26 , CD34, CD106, CD117 and CD271 in the expression profiles of surface antigen markers. In addition, the cells contained in the dental pulp-derived cell preparations are negative for at least one of, for example, all of CD19, CD26, CD34, CD56, CD106, CD117, CD146 and CD271. Among them, although the function of CD106 is not well understood, CD106 is believed to be involved in the activation of inflammatory signals because it is expressed in vascular endothelial cells at the site of inflammation. The expression profiles of these surface antigens differ from those commonly known as high colony-forming stem cells, but it is nevertheless surprising that intermediate cells and cells contained in dental pulp-derived cell preparations are highly colony-forming.

In one embodiment, when intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one, e.g., all of CD140b and HLA-A, -B and -C in expression profiles of surface antigen markers.

In one embodiment, when cells contained in dental pulp-derived cell preparations are observed as a whole, the cells are positive for at least one, e.g., all of CD49e and CD95 in surface antigen marker expression profiles.

In one embodiment, when intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole, these cells are positive for CD10 in the expression profile of surface antigen markers. Although the function of CD10 is not well understood, there is a possibility that CD10 may be involved in the convergence of inflammatory responses by degrading inflammation-associated peptides and inflammation-associated substances such as enkephalin or substance P.

Furthermore, in one embodiment, when the cells contained in the dental pulp-derived cell preparations are observed as a whole, the cells have a high CD39 positivity in the surface antigen marker expression profile compared to intermediate cells. In addition, all of these cells are positive for CD73. CD39 interacts with CD73 to convert ATP to adenosine, thereby increasing the extracellular concentration of adenosine. Adenosine suppresses inflammation by negatively controlling the immune response. It is believed that this function in the cell preparation obtained from the dental pulp, which has a percentage positive for CD39, is higher than in the intermediate cells.

In addition, in one embodiment, when intermediate cells and cells contained in dental pulp-derived cell preparations are observed as a whole (population), these cells are positive for CD46 in the expression profile of surface antigen markers. CD46 is believed to be involved in avoiding complement-dependent cytotoxicity by downregulating complement (C3) activity.

In addition, in one embodiment, when these cells are observed as a whole, these cells are positive for CD47 in the expression profile of surface antigen markers. CD47 renders cells resistant to macrophage phagocytosis.

In addition, in one embodiment, when these cells are observed as a whole, these cells are positive for CD55 in the expression profile of surface antigen markers. CD55 is thought to regulate the classical or second complement pathway and is involved in the prevention of cytotoxicity via complement.

In addition, in one embodiment, when these cells are observed as a whole, these cells are positive for CD58 in the expression profile of surface antigen markers. Although the function of CD58 is not well understood, it is suggested that CD58 promotes the convergence of inflammatory responses by inducing regulatory T cells (Treg cells).

In addition, in one embodiment, these cells are positive for CD59. CD59 is believed to be involved in the prevention of cytotoxicity due to complement by inhibiting the formation of membrane damaging complexes by acting on complement (C9).

Intermediate cells and cells contained in cell preparations derived from dental pulp secrete various humoral factors. In one embodiment, intermediate cells and cells contained in cell preparations derived from dental pulp secrete at least one, for example, all of MMP-2, IGFBP-4, cystatin C, IL-6, IL-11, MCP-1, IL-8, HGF, vascular endothelial cell growth factor (VEGF), TIMP-1, TIMP-2 and TIMP-3. In addition, intermediate cells and cells contained in cell preparations derived from dental pulp secrete at least one, for example, all of GROα, VCAM-I and IP-10.

In one embodiment, intermediate cells and cells contained in cell preparations derived from dental pulp secrete IL-6. The amount of IL-6 secreted increases due to TNF-α and IFN-γ stimulation. Although IL-6 is also known to be an inflammatory cytokine, the inflammatory response in the liver is known to be increased in IL-6 deficient mice. Therefore, IL-6 is considered to have anti-inflammatory effects.

Intermediate cells and cells contained in cell preparations derived from dental pulp secrete various humoral factors. In one embodiment, intermediate cells and cells contained in cell preparations derived from dental pulp secrete IL-11. The amount of IL-11 secreted increases due to TNF-α stimulation and IFN-γ stimulation. IL-11 is believed to exert an anti-inflammatory effect by inhibiting the secretion of an inflammatory cytokine.

Intermediate cells and cells contained in cell preparations derived from dental pulp secrete various humoral factors. In one embodiment, intermediate cells and cells contained in cell preparations derived from dental pulp secrete IP-10. The amount of IP-10 secreted increases due to TNF-α stimulation and IFN-γ stimulation. IP-10 is believed to exert an anti-inflammatory effect by inhibiting the secretion of an inflammatory cytokine.

Cells derived from dental pulp have functions such as anti-inflammatory effects. Some or all of the surface antigens and humoral factors described above, as well as other elements, interact with each other to perform their functions.

The pharmaceutical composition containing the dental pulp-derived cell preparation of the present invention as an active ingredient can be used as therapeutic agents for various diseases. For example, a cell preparation derived from dental pulp can be used to treat and prevent diseases or disorders selected from the group consisting of autoimmune diseases, inflammatory diseases, rheumatoid arthritis, Crohn's disease, chronic inflammatory bowel disease, myocardial infarction, cerebral infarction ( including chronic cerebral infarction and acute cerebral infarction), chronic inflammatory demyelinating polyneuritis, multiple sclerosis, systemic lupus erythematosus, liver cirrhosis (including decompensated liver cirrhosis), sepsis, osteoarthritis, psoriasis, and organ transplant rejection. A cell preparation containing stem cells such as mesenchymal stem cells is known as a cell preparation that can be used as a therapeutic agent for autoimmune diseases and the like. However, known cell preparations do not show significant therapeutic effects in all patients, and their use is also limited. The cell preparation obtained from dental pulp of the present invention adds variety to cell preparations that can be used as therapeutic agents for autoimmune diseases and the like. For example, new treatment options can be provided for patients who have not received any therapeutic effects from well-known cell drugs, or for patients suffering from diseases for which a therapeutic effect has not been proven with well-known cell drugs, using a cell drug derived from dental pulp of the present invention as therapeutic agents for patients.

The cell preparation derived from the dental pulp is administered to patients suffering from the diseases described above by means such as intravenous drip infusion or local injection.

The cell preparation obtained from the dental pulp is thawed during use. During application, the cell preparation obtained from the dental pulp is removed from the storage container in liquid nitrogen and thawed. Defrosting is carried out by heating in a water bath, for example from 36.5°C to 37.5°C. The thawed cell preparation obtained from dental pulp can be administered to humans as pharmaceutical products by means such as intravenous drip infusion or local injection. In the case of intravenous drip infusion, the cell preparation obtained from the dental pulp is transferred into a dialysis bag from a vial and then administered to the patient via intravenous drip infusion. In the case of local injection, the cell preparation obtained from the dental pulp is transferred into a syringe from a vial and then injected topically into the patient.

It is expected that the thawing of the cell preparation obtained from the dental pulp before use will be carried out in medical institutions. The supply of a cell preparation obtained from the dental pulp to medical institutions is carried out, for example, as follows, but the present invention is not specifically limited to this. Cellular preparations obtained from dental pulp that have been cryopreserved in the storage facilities of their manufacturers, distributors, etc., are sent in a frozen state in response to requests from medical institutions and transported to medical institutions. The preparations that have been stored in a frozen state in medical facilities are thawed immediately before administration to patients, and then administered to patients by intravenous injection or the like. A mobile low temperature worktable (WO2017/099105) can be suitably used as a device for handling frozen dental pulp-derived cell preparations.

Examples

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not intended to be limited by examples.

Example 1 Preparation of Medium and Reagent

DMEM medium (10% FBS): Prepared by adding FBS (GE Healthcare) to low glucose DMEM (at a glucose concentration of 5.56 mM, GE Healthcare) such that the final concentration of FBS was 10%.

DMEM medium (20% FBS): prepared by adding FBS (GE Healthcare) to low glucose DMEM (at 5.56 mM glucose concentration, GE Healthcare) such that the final concentration of FBS became 20%.

Streptomycin aqueous solution: Prepared by dissolving streptomycin sulfate in pure water so that the final concentration of streptomycin sulfate is 0.01 g/ml.

Protease Solution: Prepared by adding low glucose DMEM to 5 mg of liberase (Roche) so that the final concentration of low glucose DMEM is 2.5 mg/mL. It is a solution containing collagenase I, collagenase II and thermolysin as proteases.

Streptomycin DMEM (20% FBS): Prepared by mixing DMEM (20% FBS) with streptomycin solution in a 100:1 volume ratio.

Trypsin-EDTA solution: 0.25% trypsin-EDTA solution (Thermo Fisher Scientific Inc.)

1% Chlorhexidine Gluconate Solution: Prepared by diluting a 5% (w/v) solution of Fermajin (SIOE PHARMACEUTICAL CO., LTD.) 5 times with water for injection.

Ringer's Bicarbonate Solution: BICARBON infusion solution (AY Pharmaceuticals Co., Ltd.) having the composition shown in Tables 7 and 8 below.

Table 7. Component composition of ringer's bicarbonate solution

Component Concentration (g/l) Concentration (mM) Sodium chloride 6.14 105 potassium chloride 0.3 4.02 Calcium chloride dihydrochloride 0.22 1.5 Magnesium chloride hexahydrate 0.102 0.501 sodium bicarbonate 2.1 25 Sodium citrate dihydrate 0.49 1.67

Ratio of osmotic pressure of intermediate cell cryopreservation liquid to saline at pH 6.8 to 7.8 0.9 to 1.0

Table 8. Electrolyte Concentration in Bicarbonate Ringer's Solution

Electrolyte Concentration (meq/l) Na+ 135 K+ 4 Ca2+ 3 Mg2+ 1 Cl- 113 HCO3- 25 Citrate3- 5

Human serum albumin solution: KENKETSU Albumin 25-NICHIYAKU (NIHON PHARMACEUTICAL CO., LTD.) having the composition shown in Table 9 below.

Table 9. Component composition of human serum albumin solution

Component Concentration (mg/ml) Concentration (mM) Human serum albumin 250 - Acetyltryptophan sodium 5.472 20.3 sodium caprylate 3.395 20.4

(Note) Sodium ion concentration in solution: 40.7 meq/L.

Wash solution: prepared by adding 50 ml of 25% (w/v) human serum albumin solution (NIHON PHARMACEUTICAL CO., LTD.) to 1000 ml of Ringer's bicarbonate solution (BICARBON infusion solution, AY Pharmaceuticals Co., Ltd.) so so that the final concentration of human serum albumin was 1.19% (w/v). The composition of the wash solution is presented in Tables 10 and 11.

Table 10. Component composition of the wash solution

Component Concentration (g/l) Concentration (mM) Sodium chloride 5.85 100 potassium chloride 0.286 3.83 Calcium chloride dihydrochloride 0.21 1.43 Magnesium chloride hexahydrate 0.097 0.477 sodium bicarbonate 2.00 23.8 Sodium citrate dihydrate 0.467 1.59 Human serum albumin 11.9 - Acetyltryptophan sodium 0.261 0.967 sodium caprylate 0.162 0.971

Table 11. Concentration of the electrolyte contained in the wash solution (without albumin)

Component Concentration (meq/l) Na+ 130.5 K+ 3.83 Ca2+ 2.86 Mg2+ 0.952 Cl- 107.6 HCO3- 23, 8 Citrate3- 4.76 Acetyltryptophan sodium 0.967 sodium caprylate 0.971

Cytoprotective solution: prepared with Ringer's solution (BICARBON infusion solution, AY Pharmaceuticals Co., Ltd.), 25% human serum albumin solution, and dimethyl sulfoxide.

Table 12. Concentration of cytoprotective solution

Component mm Sodium chloride 46.2 potassium chloride 1.77 calcium chloride dihydrochloride 0.66 magnesium chloride 0.22 hexahydrate sodium bicarbonate eleven sodium citrate 0.73 dihydrate human serum (90) albumen Acetyltryptophan sodium 7.31 sodium caprylate 7.34 DMSO (20)

(Note) The concentration unit of human serum albumin is g/L, the concentration unit of DMSO is % (V/V).

Example 2: Obtaining dental pulp from an extracted human tooth

One human extract (third molar), obtained by informed consent, was lightly rinsed with Ringer's bicarbonate solution (BICARBON Injection) and then the surface of the extracted tooth was sterilized with 1% chlorhexidine gluconate solution. The extracted tooth was then destroyed to separate the pulp of the tooth from other tissues.

Example 3 Enzymatic Treatment of Dental Pulp

After adding the protease solution diluted with about 10 times lower low glucose DMEM to the dental pulp obtained in Example 2, the dental pulp was left in a water bath set at 37°C until it was visually observed that the tissues spread out enough.

Then, the final number of cells after the enzymatic treatment was transferred to a centrifuge tube, DMEM with streptomycin (20% FBS) was added to it to stop the enzymatic reaction, and the resulting product was centrifuged to precipitate tissue pieces, cells, etc. The supernatant was removed, DMEM with streptomycin (20% FBS) was added to the resulting pellet to suspend tissue pieces, cells, etc., and the suspension was centrifuged again to pellet the cells, etc. DMEM with streptomycin (20% FBS) was added to the pellet, and the cells were carefully suspended by pipetting to obtain a dental pulp suspension containing tissue pieces, cells derived from dental pulp, and the like.

Example 4 Dental Pulp Suspension Culture

The dental pulp suspension prepared in Example 3 was added to the cell culture plate to start the culture at 37° C. in the presence of 5% CO2. Cultivation was continued until a colony formed in the plate was visually observed by changing the DMEM medium with streptomycin (20% FBS) every 2-4 days.

Example 5 Culture of Cells Derived from Dental Pulp

The medium was removed from the cell culture dish in which the colony was observed visually, trypsin-EDTA solution was added to the dish so that the cell surfaces were covered with the solution, and the dish was allowed to stand for 5 to 10 minutes at 37°C, and the cells were peeled off. DMEM (10% FBS) was then added to the cell culture plate to stop the reaction and the cells were suspended. This cell suspension was collected in a centrifuge tube. The collected cells were centrifuged (300×g, 5 minutes) and besieged, and the supernatant was removed. DMEM medium (10% FBS) was added to a centrifuge tube and the cells were suspended to obtain a cell suspension.

After measuring the number of live cells contained in the cell suspension, the cells were seeded in a cell culture plate so that their density was from 3000 to 20000 cells/cm ² and culture was started at 37°C in the presence of 5% CO2. Cultivation was continued until the cells became confluent in the plate, replacing the medium with DMEM (10% FBS) every 2-4 days.

After removing the medium from the cell culture plate on which the cells became confluent, trypsin-EDTA solution was added to the plate so that the surface of the cells was covered with the solution. The tablet was allowed to stand for 5 to 10 minutes at 37°C to exfoliate the cells. The same amount of DMEM (10% FBS) as the trypsin-EDTA solution was then added to the plate to stop the reaction and the cells were suspended. This cell suspension was collected in a centrifuge tube. The harvested cells were centrifuged (300×g, 5 minutes) and pelleted to remove the supernatant, and DMEM (10% FBS) was added to them to suspend the cells.

The above cell collection and cultivation was repeated, and the cells proliferated until the number of living cells became 1×10 ⁸ or more. The number of cell divisions (ie, the number of divisions in the in vitro medium) during this culture period was 16 and 17, and the average cell doubling time was within 2 days (48 hours) throughout the culture period.

Example 6 Collection of Cells Derived from Dental Pulp (Intermediate Cells)

The medium was removed from the cell culture plate on which the cells became confluent in the last culture in Example 5, and the trypsin-EDTA solution was added to the plate so that the cell surfaces were covered with the solution. The tablet was allowed to stand for 5 to 10 minutes at 37°C to exfoliate the cells. The same amount of DMEM (10% FBS) as the trypsin-EDTA solution was then added to the cell culture plate to stop the reaction and suspend the cells. This cell suspension was collected in a container and centrifuged to pellet the cells, and the supernatant was removed. 500 ml to 1000 ml of cell washing solution was added to the container, and the cells were suspended. Thereafter, the suspension was again centrifuged to pellet the cells, and the supernatant was removed. This cell washing operation was repeated, and finally the cells were collected as a pellet after centrifugation. The cells thus obtained were considered as intermediate cells.

Example 7 Freezing Cells Derived from Dental Pulp (Intermediate Cells)

Wash solution and cytoprotective solution were added to the precipitated cells obtained in Example 6, and the cells were suspended so that the cell concentration was 11 x 10 ⁶ cells/ml (+5%). A cryopreservation vial (material: cyclic olefin copolymer, sterile closed vial, Aseptic Technologies) was filled with the cell suspension thus obtained and sealed. This cell suspension was considered as an intermediate cell suspension. The components contained in the solution part (intermediate cell cryopreservation liquid) of the intermediate cell suspension are shown in Table 13. The intermediate cell suspension was frozen using a programmable freezer, and then transferred to a liquid nitrogen storage container and stored therein. This frozen intermediate cell suspension was considered as a frozen intermediate cell product.

Table 13 Component composition of liquid for cryopreservation for intermediate cells

Component mM (range) mm (suitable example) Sodium chloride 65.8–80.4 73.1 potassium chloride 2.52–3.08 2.80 calcium chloride dihydrochloride 0.95–1.16 1.05 magnesium chloride 0.315–0.385 0.35 hexahydrate sodium bicarbonate 15.67–19.15 17.41 sodium citrate 1.04–1.28 1.16 dihydrate human serum (46–56) (51) albumen Sodium 3.73–4.55 4.14 acetyltryptophan sodium caprylate 3.74–4.58 4.16 DMSO (9–11) (10)

(Note) The concentration unit of human serum albumin is g/L, the concentration unit of DMSO is % (V/V).

Example 8: Thawing of Cells Derived from Dental Pulp (Intermediate Cells)

The vial filled with cells derived from dental pulp (intermediate cells) obtained in Example 7 was removed from the liquid nitrogen storage container and thawed by heating in a water bath at a temperature of 36.5°C to 37.5°C. The thawed cell suspension was transferred to a centrifuge tube and 30 ml of DMEM (10% FBS) was added to it to suspend the cell suspension. The cells were centrifuged (300×g, 5 minutes) and pelleted and the supernatant was removed. Then, 20 ml of DMEM medium (10% FBS) was added to them to suspend the intermediate cells.

Example 9 Producing Culture (Preculture) of Cells Derived from Dental Pulp

After measuring the number of intermediate living cells contained in the cell suspension prepared in Example 8, cells were seeded in a cell culture plate so that they had a density of 20,000 cells/cm ² , and culture was started at 37°C in the presence of 5% CO2. Culture was continued until the cells became confluent in the cell culture plate, replacing the medium with DMEM (10% FBS) every 2-4 days. The viability (number of living cells/number of total cells x 100%) of the cells contained in the thawed intermediate cells, which was calculated from the number of living cells, was approximately 85% to 95%.

The medium in the cell culture plate in which the cells had become confluent was removed, the trypsin-EDTA solution was added to the plate so that the cell surfaces were covered with the solution, and the plate was allowed to stand for 5 to 60 minutes at 37°C to peel off the cells. The same amount of DMEM (10% FBS) as the trypsin-EDTA solution was then added to the cell culture plate to stop the reaction and suspend the cells. The cell suspension was collected in a container and centrifuged to pellet the cells, and the supernatant was removed. DMEM medium (10% FBS) was added to them to suspend the cells, then the suspension was again centrifuged to pellet the cells and the supernatant was removed. DMEM (10% FBS) was added to them to resuspend the cells.

After measuring the number of live cells contained in the cell suspension, the cells were seeded in a cell culture plate so that their density was less than or equal to 20,000 cells/cm ² and culture was started at 37°C in the presence of 5% CO2. Cultivation was continued until the cells became confluent in the plate, replacing the medium with DMEM (10% FBS) every 2-4 days.

The collection and cultivation of cells described above was repeated, and the cells proliferated until the number of living cells became 1×10 ⁹ or more. The number of cell divisions during this culture period of these intermediate cells in the cell culture plate was 5 and 6, and the average cell doubling time was within 2 days (48 hours) throughout the culture period.

The medium in the cell culture plate in which the cells had become confluent was removed, and a trypsin-EDTA solution was added thereto. The tablet was allowed to stand for 5 to 60 minutes at 37°C to exfoliate the cells. DMEM (10% FBS) was then added to the cell culture plate to stop the reaction and the cells were suspended. This cell suspension was collected in a container. The harvested cells were centrifuged and pelleted, and the supernatant was removed. 500 ml to 600 ml of DMEM medium (10% FBS) was added thereto to suspend the cells, then the suspension was again centrifuged to pellet the cells and the supernatant was removed. 500 ml to 600 ml of DMEM (10% FBS) were added to these to resuspend the cells.

Example 10 Production Culture of Cells Derived from Dental Pulp (Bioreactor Preparation)

50 g (± 1.0 g) microcarriers were weighed using an electronic balance and placed in a reagent bottle, 2000 ml PBS was added to them, and then sterilized in an autoclave. The microcarriers used were gelatin cell culture microcarriers, which have a particle diameter of 130 to 180 μm (during hydration) and are thermally stable so that the microcarriers can be subjected to sterilization autoclaving.

A 50 L reactor bag, vent filter and sensor loop were installed in the bioreactor, and a temperature sensor, pH meter and dissolved oxygen meter were additionally installed. 10 L of DMEM (10% FBS) and 50 g (dry weight) of microcarriers were added to the (bag) bioreactor and the bioreactor was heated to 37° C. while stirring the microcarriers. Stirring was carried out using a stirrer provided in the device.

Example 11 Production Culture of Cells Derived from Dental Pulp (Stage of Cell Adhesion on Microcarriers)

After measuring the number of live cells contained in the cell suspension prepared in Example 9, the cells were added to the bioreactor so that their density was from 1000 to 5000 cells/cm3. After stirring inside the reactor for several minutes, stirring was stopped and the reactor was left for 1 hour or more. This mixing and incubation was repeated in order for the cells to attach to the microcarriers.

Example 12 Production Culture (Cell Culture Step) of Cells Derived from Dental Pulp

After completion of the adhesion step, cultivation was started with stirring inside the bioreactor. What happens inside the bioreactor was periodically observed during cultivation. In the case where the microcarriers settled at the bottom of the reactor, the stirrer speed in the reactor was increased, causing the microcarriers to float in the medium.

The production culture was continued until the cells proliferated until the number of living cells became 5×10 ⁹ or more according to the measurements described in Example 13. The number of cell divisions during the culture period on microcarriers was 2 and 3, and the average cell doubling time was approximately 3 to 6 days. Cells subjected to production culture underwent at least 23 cell divisions in the in vitro medium and 23 to 27 cell divisions.

Example 13 Production Culture of Cells Derived from Dental Pulp (Cell Number Measurement)

30 to 40 ml of the culture solution containing the microcarriers was collected from the reactor, transferred to a centrifuge tube, and left for 5 minutes or longer to allow the microcarriers to settle, and then the supernatant was removed. 25 ml of PBS was added thereto to suspend the cells, and the suspension was allowed to stand for 5 minutes or longer to remove the supernatant. This operation was carried out again. After removing the supernatant, trypsin-EDTA solution was added to the microcarriers. After shaking the mixture in a water bath at 37°C for 5-10 minutes, the same amount of DMEM medium (10% FBS) as the trypsin-EDTA solution was added to it to stop the reaction and suspend the cells. This cell suspension was centrifuged (300×g, 5 minutes) to pellet the cells and remove the supernatant. To them, 1 to 5 ml of DMEM (10% FBS) was added to suspend the cells, and then the number of live cells contained in the cell suspension was measured.

Example 14 Production Culture of Cells Derived from Dental Pulp (Measurement of Glucose Concentration and Lactic Acid Concentration)

5-40 ml of the cell suspension in culture was collected from the reactor, transferred to a centrifuge tube and allowed to stand for 5 minutes or longer to allow the microcarriers to settle. Then part of the supernatant was transferred to a new 1.5 ml tube. 50 µl of the supernatant was collected with a microsyringe, and the concentrations of glucose and lactic acids in the supernatant were measured with a biosensor (BF-7D, Oji Scientific Instruments). In the case where the glucose concentration was low, glucose was added to the medium, or all or part of the medium was replaced so that the glucose concentration did not fall below 0.1 mM. In addition, in the case where the concentration of lactic acids increased, all or part of the medium was replaced so that the concentration of lactic acids did not exceed 20 mM.

Example 15 Collection of Cells Derived from Dental Pulp Obtained in Production Culture

After confirming that the cells proliferate until the number of cells reaches the target level, mixing of the reactor and sensor loop is stopped and the cell suspension is allowed to stand for 10 minutes or longer to allow the microcarriers to settle. After removing as much of the supernatant as possible, the cells were suspended in the remaining medium. The cell suspension was collected in a 10 L bag (Thermo Fisher Scientific Inc.) and left for 5 minutes or longer to allow the microcarriers to settle and then the supernatant was removed.

Low glucose DMEM was added thereto to suspend the microcarriers, the suspension was allowed to stand for 5 minutes or longer to allow the microcarriers to settle, and then the supernatant was removed. This operation was repeated several times. Subsequently, after removal of the supernatant, trypsin-EDTA solution was added to the microcarriers. After the trypsin-EDTA solution was added thereto, the mixture was shaken in a water bath at 37° C. for 30-60 minutes to separate the cells from the microcarriers and destroy the microcarriers.

To remove fragments, cell aggregates, etc. from the microcarriers remaining in the cell suspension, the collected filtrate was passed through a filter with a pore diameter of 20 to 35 μm to collect cells from the filter filtrate. The collected filtrate was centrifuged to pellet the cells and the supernatant was removed. The washing solution prepared in Example 1 was added thereto to wash the cells, and the cells were suspended. The suspension was again centrifuged to pellet the cells and the supernatant was removed. This washing operation was repeated, and finally the cells were collected as pellets after centrifugation.

Example 16 Preparation and Cryopreservation of Cells Derived from Dental Pulp

Wash solution and cytoprotective solution were added to the precipitated cells collected in Example 15, and the cells were suspended so that the cell concentration was 25 to 31 x 10 ⁶ cells/ml.

A cryopreservation vial (material: cyclic olefin copolymer, sterile closed vial, Aseptic Technologies) was filled with the cell suspension thus obtained and sealed. This cell suspension was considered as a pre-frozen cell preparation obtained from dental pulp. The components contained in a part of the solution (cell cryopreservation liquid for cell preparation) of the pre-frozen cell preparation prepared from dental pulp are shown in Table 14.

Table 14. Component composition of cryopreservation fluid for cell preparation

Component mM (range) mm (suitable example) Sodium chloride 65.8–80.4 70.8 potassium chloride 2.52–3.08 2.71 Calcium chloride dihydrochloride 0.95–1.16 1.01 magnesium chloride 0.315–0.385 0.34 hexahydrate sodium bicarbonate 15.67–19.15 16.9 sodium citrate 1.04–1.28 1.13 dihydrate human serum (46–56) (53.6) albumen Acetyltryptophan sodium 3.73–4.55 4.36 sodium caprylate From 3.74–4.58 4.37 DMSO (9–11) (10.7)

(Note) The concentration unit of human serum albumin is g/L, the concentration unit of DMSO is % (V/V).

The pre-frozen dental pulp-derived cell preparation with which the vial was filled was frozen using a programmable freezer in the usual manner, and then transferred to a liquid nitrogen storage container. This frozen cell suspension was considered as a preparation (preparation from dental pulp cells) containing cells derived from dental pulp as active components.

Example 17 Properties (such as cell morphology) of cells derived from dental pulp)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed, 10 ml of DMEM (10% FBS) were added to them, and each suspension was gently shaken. Thereafter, each suspension was centrifuged (1500 rpm, 5 minutes) to pellet the cells. The supernatant was removed, 10 ml of DMEM (10% FBS) was added to it to suspend the cells, then the suspension was again centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 10,000 cells/cm ² , and cultured so that they became 90% to 100% confluent. Cell morphology in the cell culture plate was observed using a phase contrast microscope. All of the cultured cells contained in the frozen intermediate cell product and the pulp-derived cell preparation were essentially spindle-shaped attached cells and did not include cells with other shapes. The viability (number of living cells/number of all cells x 100%) of the cells contained in the thawed frozen intermediate cell product and the cell preparation derived from the dental pulp, calculated from the number of living cells, was approximately 85% to 95%.

Example 18: Properties (particle diameter) of a cell preparation obtained from dental pulp

The frozen intermediate cell product prepared in Example 7 and the dental pulp-derived cell preparation prepared in Example 16 were thawed and respectively diluted with D-PBS(-) solutions to obtain cell-diluted solutions at a concentration of about 5× ¹⁰⁶ cells/ml. Cell particle diameter was measured by an image analysis method using an automated cell viability analyzer (Vi-Cell XR, Beckman Coulter Inc.). An image analysis method using an automatic cell viability analyzer will be described below. Trypan blue is added to the dilute cell solution to stain the cells, this dilute cell solution is directed through the elongated flow path, and a plurality of magnified images of the flow path are photographed under a microscope. Because trypan blue permeates cell membranes of dead cells and stains dead cells, cells seen as colored particles in images are measured as dead cells, and cells seen as clear particles are measured as live cells. The cell particle diameter is calculated using the particle diameter measured in the image and the magnification of the microscope. The average value is obtained from the values of the particle diameters in all photographed images.

(Results)

The mean cell particle diameters (cell diameters) of the thawed frozen intermediate cell product were 17.36 µm ± 1.40 µm (mean ± SD, n = 3), and the mean cell particle diameters of the thawed pulp-derived cell preparation , was 16.98 ± 1.23 µm (mean ± standard deviation, n = 3) (Fig. 1).

Example 19 Properties (Ability to Differentiate into Osteocytes) of Cells Derived from Dental Pulp

The ability to differentiate into osteocytes was examined by the following method with reference to the disclosure and the like. Pittenger M.F., Et al., Science. 284, 143-7 (1999) and Colter DC., et al., Proc Natl Acad Sci USA. 98, 7841-5 (2001).

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² , and cultured so that they achieved a confluence of 90% to 100%. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. Cells were seeded in a 24-well cell culture plate coated with collagen I at cell density (15,000 to 25,000 cells/cm ² ) in DMEM (10% FBS) and each cultured for 3 days. Cells were divided into 2 groups. One group medium was replaced with osteocyte differentiation inducing medium, which was prepared by adding supplements and growth factors for osteocyte differentiation (LONZA KK.) containing dexamethasone, L-glutamine, ascorbate, penicillin/streptomycin, mesenchymal cell growth supplement (MCGS), and P-glycerophosphate, into basal osteocyte differentiation medium (LONZA KK.) for differentiation culture for 2–3 weeks, changing the medium every 3–4 days. This group was considered to be the group that induces osteocyte differentiation. The medium of the other group was replaced with fresh DMEM medium (10% FBS) for cultivation for 2–3 weeks, changing the medium every 3–4 days. This group was considered a control group. Cells from both the osteocyte differentiation-inducing group and the control group were cultured, washed once with PBS, 0.4 ml of 10% formic acid was added to each well, and the wells were allowed to stand for 1 hour at room temperature to release the calcium accumulated in cells, from cells. The released calcium concentration was quantified using Calcium E-Test Wako (Wako Pure Chemical Industries, Ltd.).

The measurement results are shown in FIG. 2. Both in the cells contained in the frozen intermediate cell product and in the cell preparation obtained from the dental pulp, the osteocyte differentiation-inducing groups showed a greater increase in the calcium concentration in the cells than in the control groups. These results show that the cells contained in both the frozen intermediate cell product and the pulp-derived cell preparation were differentiated into osteocytes, and show that the pulp-derived cells of the present invention have the ability to differentiate into osteocytes.

Example 20 Properties (Ability to Differentiate into Chondrocytes) of Cells Derived from Dental Pulp

The ability to differentiate into chondrocytes was examined by the method below with reference to the disclosure and the like. Kiani C., et al., Cell Res. 12 19-32 (2002) and Aung A., et al., Arthritis Rheum. 63 148-58 (2011).

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 10,000 cells/cm ² , and cultured so that they reached a confluence of 90% to 100%. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. Cells were divided into 2 groups. Cells of one group were suspended in Stem MACS (registered trademark) ChondroDiff Medium (Miltenyi Biotec) chondrocyte differentiation inducing medium (Miltenyi Biotec) at a concentration of 1 x 10 ⁶ cells/ml, and 1 ml of each suspension was plated in a low adsorption container (STEMFULL (registered trademark) brand), Sumitomo Bakelite Co., Ltd.) for forming spheroids. Cultivation for differentiation was carried out for 2–3 weeks, changing the medium every 3–4 days. This group was considered the group that induces chondrocyte differentiation. The cells of the other group were subcultured with DMEM (10% FBS), which was used as a control group.

After culturing, the cells were collected and treated with proteinase K for 30 minutes in a warm bath at 55°C to lyse the cells. Total RNA was extracted from the lysed cells with RNeasy (registered trademark) Plus Mini Kit (QIAGEN NV) to obtain a total RNA extract, and then the concentration of RNA contained in the total RNA extract was measured with an absorbance meter (DeNovix Inc.) After measurement of RNA concentration cDNA was synthesized using a QuantiTect reverse transcription kit (registered trademark) (QIAGEN NV). In addition, a PCR reaction solution was prepared, 25 ng cDNA of each group was used as a template, RT-PCR was performed under PCR conditions [(50°C/2 minutes) x 1 cycle, (95°C/10 minutes) x 1 cycle and (95°C/15 seconds, 60°C/1 minute) x 40 cycles] to amplify the aggrecan gene and the P-actin gene. An aggrecan probe (Applied Biosystems/Assay ID: Hs00153936_m1) and a P-actin probe (Applied Biosystems/4310881E), respectively, were used as PCR primers.

The measurement results are shown in FIG. 3. Both in the cells contained in the frozen intermediate cell product and in the cell preparation obtained from the dental pulp, it was found that the cycle threshold (Ct) values of aggrecan were higher in the groups inducing chondrocyte differentiation than in the control groups. These results show that the level of expression of aggrecan, which is the main molecule constituting the extracellular matrix of chondrocytes, increased in groups that induce chondrocyte differentiation, and show that both the cells contained in the frozen intermediate cell product and the cells of the pulp-derived preparation tooth, have the ability to differentiate into chondrocytes.

Example 21 Properties of Cells Derived from Dental Pulp (Measuring 1 Surface Antigens)

Cells derived from dental pulp had a spindle shape similar to human mesenchymal stem cells. Thus, using a flow cytometer, the presence or absence of expression of markers of surface antigens CD73, CD90, CD105 and CD166, for which human mesenchymal stem cells are known to be positive, and markers of surface antigens CD34 and CD45, for which, as is known, , mesenchymal stem cells are negative.

Phosphate buffered saline at pH 7.4, which contains BSA and is a powder (SIGMA-Aldrich), was dissolved in pure water and passed through a 0.45 µm filter to prepare PBS-B [0.01 M phosphate buffered saline solution (pH 7.4) containing 0.138 M sodium chloride, 0.0027 M potassium chloride and 1% (w/v) bovine serum albumin]. Human IgG (SIGMA-Aldrich) was dissolved in PBS (Life Technologies) and passed through a 0.45 µm filter to prepare a 20 mg/mL IgG solution. 2 ml of a 20 mg/ml IgG solution was added to 18 ml of PBS-B to prepare a blocking solution.

In addition, a FITC-labeled anti-human CD34 antibody (anti-CD34-FITC, Nippon Becton Dickinson Company, Ltd.) was used as an anti-CD34 antibody, a FITC-labeled anti-human CD45 antibody (anti-CD45-FITC, Beckman Coulter Inc.) was used as anti-CD45 antibody, FITC-labeled anti-human CD73 antibody (anti-CD73-FITC, Nippon Becton Dickinson Company, Ltd.) used as anti-CD73 antibody, FITC-labeled anti-human CD90 antibody (anti-CD73-FITC, Nippon Becton Dickinson Company, Ltd.) used as anti-CD90 antibody, PE labeled anti-human CD105 (anti-CD105-R-PE, Nippon Becton Dickinson Company, Ltd.) used as anti-CD105 antibody, PE labeled anti-human CD166 (anti -CD166-PE, Ancell Corporation) was used as anti-CD166 antibody, FITC labeled mouse IgG1 isotype control (anti-IgG1-FITC, Beckman Coulter Inc.) and PE labeled mouse IgG1 isotype control (IgG1-PE, Beckman Coulter Inc.) used as control antibodies.

(Cell staining method)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The supernatant was removed, 10 ml of DMEM (10% FBS) was added to them to suspend the cells, and the number of living cells was measured. Cells (1 x 10 ⁷ ) were collected in a 50 ml centrifuge tube, centrifuged (1500 rpm, 5 minutes) and pelleted, and the supernatant was removed. PBS-B was added to them to a total volume of 10 ml and the cells were suspended. The cells were then centrifuged again (1500 rpm, 5 minutes) to pellet the cells and the supernatant was removed. The cells were then suspended in 1 ml of blocking solution and left to stand on ice for 1 hour. Each antibody solution was added to nine 5 ml reaction tubes (numbers (1) to (9)), as shown in Table 15. Then, 100 µl of the cell suspension that had been suspended in the blocking solution was added to each tube, and the mixture was gently shaken and left on ice for 20 minutes to bind the antibodies contained in each antibody solution to the surface antigen marker expressed on the cell surface. Then, 3 ml of PBS-B was added to each tube and mixed, then the cells were centrifuged (1500 rpm, 5 minutes) and precipitated, and the supernatant was removed to remove antibodies that did not bind to the cells. This antibody removal operation was repeated 3 times. 400 μl PBS-B was added to each tube to suspend the cells.

Table 15. Type and amount of antibody solution added to each reaction tube

Tube number Antibody solution Amount added (µl) (1) PBS-B - (2) IgG1-FITC 20 (3) Anti-CD34-FITC 20 (4) Anti-CD45-FITC 20 (5) Anti-CD73-FITC 5 (6) Anti-CD90-FITC 2 (7) IgG1-PE 20 (8) Anti-CD105-FITC 2 (9) Anti-CD 166-FITC 20

(Measurement and analysis)

As for the cells of tubes numbered (1) to (9), the amount of each fluorescent dye from FITC and PE was measured by BD FACSVerse (registered trademark) (Nippon Becton Dickinson Company, Ltd.) to obtain the amount of fluorescent dye associated with cell surfaces through antibodies specifically bound to surface antigens compared to negative controls. Tube (2) is the negative control for tubes (3) - (6) and tube (7) is the negative control for tubes (8) and (9). Tube (1) is unstained cells.

The measurement results are shown in FIG. 4. For cells with tube numbers (5), (6), (8), and (9) (which were stained with CD73-, CD90-, CD105-, and CD166, respectively), 85% or more of the cells were positive for these surface antigens both in the cells contained in the product from frozen intermediate cells, and in the cell preparation obtained from the dental pulp. For cells with tube numbers (3) and (4) (which were cells stained for CD34 and CD45, respectively), most of the cells were negative for these surface antigens in both the cells contained in the frozen intermediate cell product and in a cell preparation obtained from the dental pulp. These results indicate that cells derived from dental pulp are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45.

Example 22 Properties of Cells Derived from Dental Pulp (Measurement of 2 Surface Antigens)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and the final amount of each cell suspension was added to DMEM medium. The cell suspension was centrifuged (300 x g, 5 minutes, room temperature) and the supernatant was removed. After that, DMEM medium was added to them to suspend the cells. Subsequently, cells were stained with antibodies against surface antigens using a BD Lyoplate (human cell marker screening panel, Nippon Becton Dickinson Company, Ltd.). Antibody staining was performed according to the protocol of the Human Cell Screening Panel (Nippon Becton Dickinson Company, Ltd.). The outline of the method for measuring surface antigens using a human cell screening panel will be described below. The cell suspension is dispensed into each well of a 96-well plate. Antibodies against various surface antigens were respectively added to the wells as primary antibodies and left for 30 minutes for the surface antigens to bind to the antibodies. The cell suspension was centrifuged to pellet the cells and the supernatant was removed. After washing the cells, the antibodies against the primary antibodies were respectively added to the wells as secondary antibodies and left for 30 minutes for the surface antigens to bind to the antibodies. The secondary antibodies were fluorescently labeled with Alexa Fluor 647. The cell suspension was centrifuged to pellet the cells and the supernatant was removed. The cells were resuspended. With respect to this cell suspension, the cells to which the secondary antibodies are bound are fluorescently detected as positive cells by flow cytometry to obtain the relative abundance of positive cells for surface antigens. Measurements were performed on 9 batches of intermediate cells and 7 batches of cells contained in a cell preparation derived from dental pulp to obtain a positive ratio for each antigen.

(Results)

The surface antigens for which intermediate cells and cells contained in a cell preparation derived from dental pulp were positive are shown respectively in FIG. 5 and 6.

In intermediate cells, positivity rates of 95% or higher were shown for CD47, CD81, CD90, CD147 and HLA-A, -B and -C in all batches. In addition, positivity rates of 90% or higher were shown for CD29, CD46, CD55, CD59, CD73, and CD140b across all lots. In addition, positivity rates of 80% or greater, approximately 90% or greater were shown for CD9, CD44, CD49b, CD49c, CD98 and EGF-R in all batches. In addition, positivity ratios of 70% or higher, approximately 90% or higher were shown for CD49f and CD166 in all cells.

In addition, positivity rates of 60% or higher, about 90% or higher were shown for CD10, CD13, CD58, CD63, CD151 and CD164 in all cells (FIG. 5). In addition to this, the intermediate cells are also positive for CD105 (FIG. 4).

On the other hand, in cells contained in a cell preparation derived from dental pulp, positivity rates of 95% or higher, approximately 98% or higher were shown for CD29, CD59, CD44 and CD164 in all batches. In addition, positivity rates of 90% or greater, approximately 95% or greater have been shown for CD9, CD13, CD46, CD47, CD58, CD63, CD73, CD81, CD90, CD98, CD147, EGF-R and HLA-A, -B and -C in all batches. In addition, positivity rates of 80% or higher have been shown for CD49b, CD49c, CD49e, CD55, CD95,

CD151 and CD166 in all lots and showed positivity rates of approximately 90% or higher, except for CD95, CD151 and CD166. In addition, positivity rates of 70% or higher, approximately 80% or higher were shown for CD10, CD49f and CD140b in all batches (FIG. 6). In addition, the cells contained in the cell preparation obtained from dental pulp are also positive for CD105 (FIG. 4).

Then, the Surface antigens for which the intermediate cells and the cells contained in the cell preparation obtained from the dental pulp were negative, respectively, are shown in Tables 16 and 17.

In intermediate cells, positivity rates of 1% or lower have been shown for CD120b, CD132, CD158a, CD161, CD184, CD195, CD206, CD210, CD212, CD226, CD244, CD267, CD278, CD279, CD282, CD294, NKB1, SSEA-1, TRA-1-60, TRA-1-81, Vp23, SSEA-3, CLA and β7 integrin in all batches. In addition, positivity rates of 2% or lower have been shown for CD8b, CD11b, CD15s, CD16, CD19, CD24, CD31, CD32, CD62E, CD62P, CD66f, CD86, CD88, CD94, CD100, CD103, CD104, CD114, CD117, CD118, CD121b, CD122, CD123, CD124, CD126, CD127, CD128b, CD135, CD137, CD137, CD150, CD163, CD172b, CD177, CD178, CD180, CD197, CD220, CD229, CD231, CD1430, CD2 CD32 1, CDw327, CDw328, CD329, CD335, CD336, BLTR-1, CLIP, CMRF-44, CMRF-56, fMLP-R, Vβ8, invariant NKT, and γδ TCR in all lots, and approximate positivity rates were 1% or lower. In addition, positivity rates of 5% or lower have been shown for CD1a, CD1b, CD1d, CD2, CD3, CD5, CD6, CD7, CD8a, CD11c, CD15, CD18, CD21, CD22, CD23, CD25, CD27, CD28, CD33. CD35, CD37, CD38, CD41a, CD41b, CD42b, CD45, CD45RB, CD45RO, CD48, CD50, CD53, CD62L, CD64, CD66 (a, c, d, e), CD69, CD70, CD72, CD74, CD84, CD85 , CD87, CD89, CDw93, CD97, CD134, CD138, CD141, CD144, CD154, CD158b, CD162, CD183, CD205, CD235a, CD309, CD326, CD337 and αβ TCR in all cells. In addition, although positivity rates greater than 5% were shown for CD26, CD106 and CD271 in some lots, the average positivity rates were 5% or lower (Table 16A - Table 16D). In addition to this, intermediate cells are also negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigen (FIGS. 4 and 11A).

In cells contained in a pulp-derived cell preparation, positivity rates of 1% or lower have been shown for CD1a, CD1d, CD2, CD3, CD4, CD5, CD7, CD8a, CD8b, CD11b, CD11c, CD15, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD28, CD30, CD31, CD32, CD33, CD34, CD35, CD37, CD38, CD41a, CD86, CD87, CD88, CD89, CDw93, CD94, CD100, CD102, CD103, CD114, CD117, CD118, CD120b, CD121b, CD122, CD123, CD124, CD126, CD127, CD128b, CD132, CD134, CD135, CD137, CD137, CD138, CD144, CD150, CD138, CD144, CD 150, CD158a , CD158b, CD161, CD162, CD163, CD172b, CD177, CD178, CD180, CD184, CD195, CD196, CD197, CD205, CD206, CD210, CD212, CD220, CD226, CD229, CD231, CD235a, CD244, CD267CD26 8, CD278, CD279, CD282, CD294, CD305, CD309, CD314, CD321, CDw327, CDw328, CD329, CD335, CD336, CD337, BLTR-1, CLIP, CMRF-44, CMRF-56, fMLP-R, SSEA-1, TRA- 1-60, CLA, β7 integrin, and invariant NKT in all batches. In addition, positivity rates of 2% or lower have been shown for CD1b, CD6, CD27, CD41b, CD42a, CD42b, CD43, CD45, CD45RB, CD48, CD50, CD53, CD57, CD62E, CD62L, CD62P, CD64, CD66b, CD66f, CD69, CD70, CD72, CD75, CD84, CD85, CD97, CD99R, CD183, CD193, SSEA-3, and γδ TCR in all lots, and the approximate positivity rates were 1% or lower. In addition, positivity rates of 5% or lower have been shown for CD4v4, CD14, CD36, CD45RA, CD45RO, CD66 (a, c, d, e), CD79b, CD83, CD152, CD209, CD275, CD326, MIC A/B, and αβ TCR in all lots, and the approximate positivity rates were 2% or lower. In addition, although positivity rates greater than 5% were shown for CD106 and CD271 in some lots, the average positivity rates were 5% or lower (Table 17A - Table 17E). In addition, the cells contained in the pulp-derived cell preparation are also negative for CD34, CD40, CD45, CD80, CD86 and MHC class II antigens (FIGS. 4 and 11B).

Table 16A. Surface antigens for which intermediate cells were negative

surface antigen Average value for intermediates SE CD1a 0.49 0.32 CD1b 1.61 0.57 CD1d 0.89 0.32 CD2 0.79 0.33 CD3 0.00 0.47 CD5 0.00 0.49 CD6 1.15 0.44 CD7 0.64 0.30 CD8a 0.74 0.34 CD8b 0.00 0.55 CD11b 0.00 0.32 CD11c 0.81 0.29 CD15 1.04 0.46 CD15s 0.58 0.18 CD16 0.44 0.25 CD18 0.67 0.37 CD19 0.25 0.32 CD21 0.65 0.38 CD22 0.67 0.32 CD23 0.7 0.29 CD24 0.00 0.36 CD25 0.86 0.33 CD26 3.49 1.18 CD27 0.90 0.48 CD28 0.88 0.44 CD31 0.25 0.32 CD32 0.17 0.37 CD33 0.57 0.37 CD35 0.61 0.39 CD37 0.58 0.32 CD38 0.91 0.41 CD41a 0.40 0.35 CD41b 0.44 0.32 CD42b 0.39 0.32 CD45 0.67 0.34 CD45RB 0.59 0.29

Table 16B. Surface antigens for which intermediate cells were negative

surface antigen Average value for intermediates SE CD45RO 0.94 0.37 CD48 0.64 0.30 CD50 0.84 0.50 CD53 0.56 0.33 CD62E 0.14 0.33 CD62L 0.33 0.31 CD62P 0.14 0.19 CD64 0.27 0.33 CD66 (a, c, d, e) 0.06 0.40 CD66f 0.17 0.32 CD69 0.40 0.45 CD70 0.17 0.33 CD72 0.17 0.38 CD74 0.00 0.66 CD84 0.42 0.39 CD85 0.02 0.45 CD86 0.23 0.21 CD87 0.66 0.46 CD88 0.30 0.19 CD89 0.56 0.32 CDw93 0.42 0.40 CD94 0.38 0.23 CD97 0.67 0.27 CD100 0.28 0.24 CD103 0.24 0.19 CD104 0.22 0.19 CD106 4.82 1.68 CD114 0.12 0.18 CD117 0.38 0.23 CD118 0.25 0.17 CD120b 0.04 0.05 CD121b 0.13 0.19 CD122 0.20 0.27 CD123 0.21 0.18 CD124 0.21 0.25 CD126 0.12 0.20

Table 16C. Surface antigens for which intermediate cells were negative

surface antigen Average value for intermediates SE CD127 0.23 0.26 CD128b 0.09 0.19 CD132 0.19 0.05 CD134 0.26 0.33 CD135 0.38 0.23 CD137 0.22 0.26 Ligand CD137 0.34 0.25 CD138 0.54 0.38 CD141 0.81 0.37 CD144 0.29 0.42 CD150 0.18 0.18 CD154 0.26 0.26 CD158a 0.00 0.09 CD158b 0.02 0.32 CD161 0.02 0.20 CD162 0.20 0.32 CD163 0.00 0.19 CD172b 0.21 0.25 CD177 0.03 0.21 CD178 0.06 0.23 CD178 0.16 0.27 CD183 1.24 0.51 CD184 0.00 0.27 CD195 0.00 0.27 CD197 0.36 0.17 CD205 0.76 0.53 CD206 0.00 0.17 CD210 0.18 0.03 CD212 0.31 0.08 CD220 0.03 0.19 CD226 0.00 0.16 CD229 0.12 0.20 CD231 0.00 0.20 CD235a 0.77 0.50 CD244 0.00 0.43 CD255 0.16 0.18

Table 16D. Surface antigens for which intermediate cells were negative

surface antigen Average value for intermediates SE CD267 0.20 0.05 CD268 0.19 0.18 CD271 3.62 0.74 CD278 0.00 0.10 CD279 0.17 0.17 CD282 0.19 0.15 CD294 0.06 0.05 CD305 0.33 0.21 CD309 0.39 0.26 CD314 0.35 0.21 CD321 0.40 0.25 CD326 2.37 0.41 CDw327 0.34 0.22 CDw328 0.42 0.22 CD329 0.37 0.18 CD335 0.31 0.18 CD336 0.20 0.18 CD337 0.84 0.42 Αβ TCR 1.44 0.27 BLTR-1 0.33 0.22 CLIP 0.25 0.23 CMRF-44 0.48 0.23 CMRF-56 0.26 0.23 fMLP-R 0.44 0.20 γδTCR 0.48 0.19 Invariant NKT 0.08 0.15 NKB1 0.22 0.12 SSEA-1 0.00 0.15 TRA-1-60 0.00 0.12 TRA-1-81 0.00 0.10 Vp23 0.21 0.13 Vp8 0.44 0.15 SSEA-3 0.11 0.05 CLA 0.07 0.05 Integrin β7 0.06 0.04

Table 17A. Surface antigens for which the cells contained in the preparation were negative

surface antigen Product average SE CD1a 0.00 0.85 CD1b 0.00 0.80 CD1d 0.00 0.83 CD2 0.00 0.73 CD3 0.00 0.22 CD4 0.00 0.76 CD4v4 0.52 0.84 CD5 0.00 0.24 CD6 0.00 0.78 CD7 0.00 0.77 CD8a 0.00 0.85 CD8b 0.00 0.17 CD11b 0.00 0.23 CD11c 0.00 0.80 CD14 0.71 0.36 CD15 0.05 0.21 CD15s 0.00 0.21 CD16 0.00 0.84 CD18 0.00 0.82 CD19 0.00 0.75 CD20 0.00 0.22 CD21 0.00 0.75 CD22 0.00 0.76 CD23 0.00 0.80 CD24 0.00 0.24 CD25 0.00 0.78 CD26 9.03 1.86 CD27 0.00 0.78 CD28 0.00 0.81 CD30 0.00 0.74 CD31 0.00 0.69 CD32 0.00 0.21 CD33 0.00 0.71

Table 17B. Surface antigens for which the cells contained in the preparation were negative

surface antigen Product average SE CD34 0.00 0.58 CD35 0.00 0.75 CD36 0.71 0.28 CD37 0.00 0.75 CD38 0.00 0.74 CD41a 0.00 0.75 CD41b 0.00 0.29 CD42a 0.00 0.69 CD42b 0.00 0.83 CD43 0.00 0.78 CD45 0.00 0.83 CD45RA 1.10 0.44 CD45RB 0.00 0.84 CD45RO 0.74 0.43 CD48 0.19 0.29 CD50 0.29 0.38 CD53 0.00 0.73 CD56 2.67 1.53 CD57 0.13 0.29 CD62E 0.00 0.75 CD62L 0.00 0.74 CD62P 0.00 0.79 CD64 0.00 0.85 CD66 (a, c, d, e) 0.14 0.37 CD66b 0.43 0.33 CD66f 0.00 0.84 CD69 0.00 0.84 CD70 0.00 0.31 CD72 0.00 0.29 CD75 0.01 0.33 CD79b 0.91 0.93 CD83 0.51 0.86 CD84 0.00 0.73

Table 17C. Surface antigens for which the cells contained in the preparation were negative

CD85 0.00 0.35 CD86 0.00 0.77 CD87 0.00 0.79 CD88 0.00 0.82 CD89 0.00 0.78 CDw93 0.00 0.24 CD94 0.00 0.76 CD97 0.00 0.90 CD99R 0.00 0.30 CD100 0.00 0.77 CD102 0.00 0.20 CD103 0.00 0.80 CD106 4.43 0.79 CD114 0.00 0.77 CD117 0.00 0.72 CD118 0.00 0.82 CD120b 0.00 0.07 CD121b 0.00 0.78 CD122 0.00 0.79 CD123 0.00 0.78 CD124 0.00 0.77 CD126 0.00 0.77 CD127 0.00 0.78 CD128b 0.00 0.81 CD132 0.26 0.06 CD134 0.00 0.80 CD135 0.00 0.76 CD137 0.00 0.76 Ligand CD137 0.00 0.79 CD138 0.00 0.76 CD144 0.00 0.79 CD146 9.80 3.52 CD150 0.00 0.80

Table 17D. Surface antigens for which the cells contained in the preparation were negative

surface antigen Product average SE CD152 0.35 0.50 CD153 0.00 0.74 CD154 0.00 0.73 CD158a 0.00 0.14 CD158b 0.00 0.23 CD161 0.00 0.77 CD162 0.00 0.74 CD163 0.00 0.78 CD172b 0.00 0.63 CD177 0.00 0.77 CD178 0.00 0.77 CD178 0.00 0.79 CD183 0.00 0.79 CD184 0.00 0.14 CD193 0.12 0.33 CD195 0.00 0.16 CD196 0.00 0.71 CD197 0.00 0.18 CD205 0.00 0.22 CD206 0.00 0.79 CD209 1.62 0.47 CD210 0.00 2.02 CD212 0.00 2.09 CD220 0.00 0.80 CD226 0.00 0.78 CD229 0.00 0.79 CD231 0.00 0.78 CD235a 0.00 0.22 CD244 0.00 0.11 CD255 0.00 0.19 CD267 0.00 2.11 CD268 0.00 0.67 CD271 4.62 0.78

Table 17E. Surface antigens for which the cells contained in the preparation were negative

CD275 0.74 0.44 CD278 0.00 0.76 CD279 0.00 0.73 CD282 0.00 0.82 CD294 0.00 2.05 CD305 0.00 0.78 CD309 0.00 0.83 CD314 0.00 0.81 CD321 0.00 0.75 CD326 0.99 0.74 CDw327 0.00 0.77 CDw328 0.00 0.82 CD329 0.00 0.83 CD335 0.00 0.81 CD336 0.00 0.81 CD337 0.00 0.74 αβ TCR 0.95 0.27 BLTR-1 0.00 0.79 CLIP 0.00 0.74 CMRF-44 0.27 0.18 CMRF-56 0.00 0.85 fMLP-R 0.00 0.82 γδTCR 0.00 0.80 Invariant NKT 0.00 0.79 SSEA-1 0.00 0.12 TRA-1-60 0.00 0.13 SSEA-3 0.96 0.21 CLA 0.14 0.13 Integrin β7 0.00 2.03 MIC A/B 0.45 0.41

Surface antigens that had a positivity ratio difference of at least 10% between intermediate cells and cells contained in a cell preparation derived from dental pulp are shown in FIG. 7. In cells contained in a pulp-derived cell preparation obtained by further culturing intermediate cells using the methods described in Examples 9-15, the positivity rates of CD39, CD49a, CD61, CD107a, CD107b and CD143 increased by 20% or more, but the CD146 positivity ratio decreased by 60% compared to intermediate cells. These results show that the cells contained in the cell preparation obtained from dental pulp have different properties from the intermediate cells. Thus, these results show that the cells contained in the cell preparation obtained from dental pulp can be obtained as new cells other than intermediate cells by culturing using the methods described in Examples 9-15.

Example 23 Properties of Cells Derived from Dental Pulp (Vessel Endothelial Cell Growth Factor (VEGF) Secretion Test)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² , and cultured so that they became 90% to 100% confluent. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. After cells were suspended in DMEM (10% FBS) at a concentration of 0.7 x 10 ⁵ cells/ml, 10 ml of each suspension was plated in a T25 cell culture plate and cultured for 5 days. After culturing, the total amount of the culture supernatant was collected, the mass of the collected culture supernatant was measured with an electronic balance (Shimadzu Corporation), and the culture supernatant was cryopreserved at -80° C. until measured. After collecting and removing the supernatant, the cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured.

The above cryopreserved culture supernatant was thawed, VEGF contained in the culture supernatant was labeled with the Quantikine ELISA Human VEGF Kit (R&D Systems) and then detected with a microplate reader (Molecular Devices, LLC). The resulting detected value was interpolated to a calibration curve generated with VEGF at a well-known concentration, and the concentration of VEGF contained in the culture supernatant was obtained. The amount of secreted VEGF was calculated as the secreted amount per number of cells according to the following equation.

[VEGF secretion amount = measured value of VEGF concentration x culture supernatant volume/number of live cells (1 x 10 ⁵ cells)]

Fig. 8 is an example of measurement results. These results show that both the intermediate cells and the cells contained in the dental pulp-derived cell preparation have the ability to secrete VEGF, but this ability is enhanced by further culture of the intermediate cells to obtain a dental pulp-derived cell preparation. Since VEGF is an angiogenic substance, the dental pulp-derived cell preparation can promote the formation of blood vessels in the body using VEGF by administering this dental pulp-derived cell preparation to humans. In addition, since the secretory ability of VEGF is enhanced in the cells contained in the dental pulp-derived cell preparation obtained through intermediate cells, it is believed that the dental pulp-derived cell preparation prepared by this preparation method is effective as a therapeutic agent for diseases in which it is necessary to exert an angiogenic effect.

Example 24 Properties of Cells Derived from Dental Pulp (Prostaglandin E2 (PGE2) Secretory Capacity Test)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² , and cultured so that they became 90% to 100% confluent. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. After cells were suspended in DMEM (10% FBS) at a concentration of 1 x 10 ⁵ cells/ml, 1 ml of each of the diluted cells were seeded into 6 wells in a 12-well cell culture plate. In addition, 1 ml of DMEM (10% FBS) containing 40 ng/ml TNF-α (R&D Systems) was added to each of the 3 wells, and 1 ml of DMEM (10% FBS) was added to each of the remaining 3 wells. The former were cultured as a TNF-α stimulated group, and the latter were cultured as a non-stimulated group (control group) for about 24 hours. After culturing, the total amount of culture supernatants was collected, the mass of the collected supernatants was measured with an electronic balance (Shimadzu Corporation), and the supernatants were cryopreserved at -80° C. until measured. After collecting and removing the supernatants, the cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured.

The above cryopreserved culture supernatant was thawed, prostaglandin E2 (PGE2) contained in the culture supernatant was labeled with the Prostaglandin E2 Express EIA kit (Cayman Chemical) and then detected with a microplate reader (Molecular Devices, LLC). The resulting detected value was interpolated to a calibration curve generated with PGE2 at a well-known concentration, and the concentration of PGE2 contained in the culture supernatant was obtained. The amount of PGE2 secreted was calculated as the secreted amount per 1 x 10 ⁵ cells according to the following equation.

[Amount of secreted PGE2 = measured value of PGE2 concentration x volume of culture supernatant/number of live cells (1 x 10 ⁵ cells)]

The measurement results are shown in Fig.9. These results show that both the intermediate cells and the cells contained in the pulp-derived cell preparation have TNFα-reactive PGE2 secretory ability, but this ability is enhanced when the intermediate cells are further cultured to obtain the pulp-derived cell preparation. . Since PGE2 has a strong anti-inflammatory effect, the dental pulp-derived cell preparation acts on the inflammatory site in the body through PGE2, and can suppress tissue destruction accompanied by inflammation by administering the cells to a human. In addition, since the secretory ability of PGE2 is enhanced in the cells contained in the dental pulp-derived cell preparation obtained through intermediate cells, it is believed that the dental pulp-derived cell preparation obtained by the preparation method is effective as a therapeutic agent in diseases in which it is necessary to exert an anti-inflammatory effect.

Example 25 Kynurenine Secretion Test

(test method)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and then plated in a cell culture flask to a density of 5,000 to 15,000 cells/cm ² and cultured for 4 days so that the cells reach 90% - 100% confluence. The cells were washed with PBS, trypsin-EDTA (Thermo Fisher Scientific Inc.) was added to them, and each mixture was allowed to stand for 5-10 minutes at 37° C. to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. Cells were seeded in two cell culture flasks so that they had a density of 15,000 to 25,000 cells/cm ² . DMEM (10% FBS) containing 100 U/ml IFN-γ (SHIONOGI Co., Ltd.) was added to one of the cell culture flasks, and DMEM (10% FBS) was added to the other cell culture flask. The former were cultured as the IFN-γ stimulated group, and the latter were cultured as the non-stimulated group (control group) for 3 days. After cultivation, the total amount of cell culture supernatants was collected. The mass of the collected cell culture supernatants was measured with an electronic balance (Shimadzu Corporation), and the supernatants were cryopreserved at -80° C. prior to measurement. After collecting and removing the supernatants, the cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added thereto to suspend the cells, and the number of living cells in each of the IFN-γ-stimulated group and the unstimulated group was measured. The above cryopreserved culture supernatant was thawed and 30% trichloroacetic acid (NACALAI TESQUE, INC.) was added thereto to perform centrifugation (10,000 g, 10 minutes). Thereafter, kynurenine contained in the culture supernatant was determined by HPLC (Shimadzu Corporation). The resulting detected value was interpolated to a calibration curve generated with kynurenine at a well-known concentration, and the concentration of kynurenine (molar concentration) contained in the culture supernatant was obtained. The amount of secreted kynurenine was calculated as the amount of secretion per number of cells according to the following equation.

[Amount of secreted kynurenine = measured value of kynurenine concentration x 208.21 x volume of culture supernatant/number of living cells (1 x 10 ⁵ cells)]

(Results)

The results are shown in Table 10. When both intermediate cells and a cell preparation derived from dental pulp are cultured in the presence of IFN-γ, the amount of kynurenine secreted increases significantly. Kynurenine can inhibit T cell proliferation and differentiate monocytes into anti-inflammatory M2 macrophages. Accordingly, it is believed that dental pulp-derived cells enriched in pluripotent stem cells can exert anti-inflammatory effects using kynurenine when administered to humans.

Example 26 Properties of Cells Derived from Dental Pulp (Low Immunogenicity Test)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² , and cultured so that they became 90% to 100% confluent. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. The cells were seeded in two cell culture plates so that their density was between 15,000 and 25,000 cells/cm ² . DMEM (10% FBS) containing 100 U/ml IFN-γ (SHIONOGI Co., Ltd.) was added to one of the cell culture plates, and DMEM (10% FBS) was added to the other cell culture plate. The former were cultured as the IFN-γ stimulated group, and the latter were cultured as the non-stimulated group (control group) for 3 days. After culturing, the cells were washed with PBS, trypsin-EDTA was added thereto, and each mixture was allowed to stand for 5-10 minutes at 37° C. to detach the cells. DMEM medium (10% FBS) was added thereto to suspend the cells, and the number of living cells in each of the IFN-γ-stimulated group and the unstimulated group was measured. Then the cell suspension of each group was centrifuged (1500 rpm, 5 minutes) and the supernatant was removed. Then, a blocking solution (described in Example 21) was added to them to bring the cell concentration to 1×10 ⁷ cells/ml, and left on ice for 1 hour.

FITC labeled anti-human MHC class I antibody (Ancell Corporation), FITC labeled anti human MHC class II antigen (Ancell Corporation), FITC labeled anti human CD40 antibody (BD Biosciences), FITC labeled anti human CD80 antibody (BD Biosciences ) and FITC-labeled anti-human CD86 antibody (BD Biosciences), respectively, were used as antibodies against MHC class I antigen, MHC class II antigen, CD40, CD80 and CD86. In addition, a FITC labeled mouse IgG1 isotype control (Beckman Coulter Inc.) and a FITC labeled mouse IgG2a isotype control (BD Biosciences) were used as control antibodies.

Each antibody solution was added to 5 ml reaction tubes (numbers (1) to (16)) as shown in Table 16. Then, 100 µl of the cell suspension of the IFN-γ stimulated group was added to each of the 5 ml reaction tubes (1 ) - (8) and added 100 μl of the cell suspension of the IFN-γ stimulated group to each of the 5 ml reaction tubes (9) - (16), and the mixture was gently shaken and left on ice for 20 minutes to bind the antibodies contained in each antibody solution, with antigen surface markers expressed on the cell surface. Then, 3 ml of PBS-B was added to each tube and mixed, then the cells were centrifuged (1500 rpm, 5 minutes) and precipitated, and the supernatant was removed to remove antibodies that did not bind to the cells. This antibody removal operation was repeated 3 times. Then, 400 μl of PBS-B was added to each tube to suspend the cells.

Table 18. Type and amount of antibody solution added to each reaction tube

Tube number Antibody solution Added quantity (19) - - (2), (10) IgG1-FITC 20 (3), (11) IgG2a-FITC 20 (4), (12) Anti-MHC-Class I 2 (5), (13) Anti-MHC-Class II 2 (6), (14) Anti-CD40-FITC 20 (7), (15) Anti-CD80-FITC 20 (8), (16) Anti-CD86-FITC 20

For tube cells (1) to (16), the amount of FITC fluorescent dye was measured using BD FACSVerse (Nippon Becton Dickinson Company, Ltd.) to obtain the amount of fluorescent dye bound to cell surfaces via antibodies specifically bound to surface antigens compared to negative controls. Tube (2) is the negative control for tubes (4) and (6) - (8), tube (3) is the negative control of tube (5), tube (10) is the negative control of tubes (12) and (14) - ( 16) and tube (11) is the negative control of tube (13). Tubes (1) and (9) are unstained cells.

(Results)

The measurement results are shown in Fig. 11A and 11B. With respect to the MHC-class I antigen measurement, approximately all cells in the intervening cells and in the pulp-derived cell preparation were positive regardless of the presence or absence of IFN-γ stimulation. With respect to MHC class II antigen measurements, all cells were essentially negative due to lack of IFN-γ stimulation, but most cells were positive due to IFN-γ stimulation in intermediate cells and pulp-derived cell preparation. With regard to the results of measuring CD40, CD80 and CD86, approximately all cells in the intermediate cells and cell preparation obtained from dental pulp were negative regardless of the presence or absence of IFN-γ stimulation.

The MHC class I antigen and the MHC class II antigen are cell surface antigens that perform the function of antigen presentation to immune cells. In addition, it is known that the MHC class I antigen is expressed in almost all cells and that the expression of the MHC class II antigen is induced in many cells by IFN-γ stimulation. On the other hand, CD40, CD80 and CD86 are surface antigens involved in the activation of cells of the immune system. That is, because the frozen intermediate cell product and the dental pulp-derived cell preparation do not express CD40, CD80, and CD86, it is believed that they do not actively activate immune cells. Thus, these results demonstrate that the cells contained in both the frozen intermediate cell product and the dental pulp-derived cell preparation are not immunogenic and therefore have the property of not inducing immunogenicity even when stimulated with IFN-γ.

Example 27: Measurement of secretory capacity of various factors including cytokines, intermediate cells and cells contained in a cell preparation obtained from dental pulp (preculture)

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and the final amount of each thawed cell suspension was added to DMEM medium. Centrifugation (300 xg, 5 minutes, room temperature) was performed and the supernatant was removed. Thereafter, DMEM was added thereto to suspend the cells, and the number of living cells and the number of total cells in the cell suspension were measured with a Muse Cell Analyzer (Millipore Sigma) to calculate viability. The cells were seeded in a T225 flask so that the density of living cells was 7000 cells/cm ² for the cells of the product from frozen intermediate cells and 12000 cells/cm ² for the cells of the cell preparation obtained from the dental pulp, and cultured in a CO2 incubator (37°C , 5% CO2) for 4 days. After cultivation, the medium was removed from the T225 flask. The cells were then washed with DPBS, 0.25% trypsin-EDTA was added to them, and each mixture was allowed to stand in a CO2 incubator for 5 minutes. After confirming cell shedding, DMEM was added to them to suspend the cells, and the total amount of each cell suspension was collected. Centrifugation (300 xg, 5 minutes, room temperature) was performed to pellet the cells. After removing each supernatant, DMEM was added thereto to suspend the cells, and the number of living cells and the number of total cells in each cell suspension were measured with a Muse Cell Analyzer (Millipore Sigma) to calculate viability.

(Collection of culture supernatant)

Cells collected in preculture were plated in T25 flasks so that the number of live cells was 28,000 cells per flask, and they were divided into an unstimulated group, a TNF-α stimulated group, and an IFN-γ stimulated group to start culturing. The amount of medium for each group was 10 ml per flask and the final concentration of TNF-α was adjusted to 10 ng/ml (using 2 μg/ml stock solution) and the final concentration of IFN-γ was adjusted to 100 U/ml (1 x 106 U/ml). On the 3rd day after the start of cultivation, 0.5 ml of the supernatant of each culture was collected in a cryovial and cryopreserved. After collecting the culture supernatants, the cells were washed with DPBS, 0.25% trypsin-EDTA was added to them, and each mixture was allowed to stand in a CO2 incubator for 5 minutes. After confirming cell shedding, DMEM medium was added to them, and the final amount of each cell suspension was collected. Centrifugation was performed (300 x g, 5 minutes, room temperature) and each supernatant was removed. Thereafter, DMEM was added thereto for cell suspension, and the number of living cells and the number of total cells in each cell suspension were measured with a Muse Cell Analyzer (Millipore Sigma) to calculate viability.

(Measurement of cytokines)

The concentrations of various factors, including cytokine, contained in the collected culture supernatants were measured using the LEGENDplex (BioLegend, Inc.). The LEGENDplex measurement principle will be described below. The primary antibody, which is bound to the granules and differs for each antigen (cytokine type), is added to the sample containing the substance to be measured for the binding of the primary antibody to that substance. Subsequently, a biotinylated secondary antibody was added thereto and bound to a substance bound to the primary antibody, and streptavidin labeled with phycoerythrin (PE) was further bound to it. Because avidin specifically binds to biotin, this can be measured using flow cytometry to quantify phycoerythrin (PE) labeled bead-bound streptavidin by its fluorescence intensity. Since this quantitative value is proportional to the amount of the measured substance contained in the sample, this substance can be quantified.

(Results: expression level of various factors in unstimulated groups)

The concentrations of the various factors contained in the culture supernatants in the unstimulated groups are shown in FIG. Both the intermediate cells and the cells contained in the pulp-derived cell preparation express MMP-2, IGFBP-4 and cystatin C at high levels (FIG. 12).

In addition, both intermediate cells and cells contained in a cell preparation obtained from dental pulp express IL-6, IL-11, MCP-1, IL-8, GROα, HGF, VEGF, VCAM-1, TIMP- 3, TIMP-2 and TIMP-1 (FIG. 13).

In addition, the number of intermediate cells and cells contained in the dental pulp-derived cell preparation is small, but both the intermediate cells and the cells contained in the dental pulp-derived cell preparation express IL-23, TNF. -α, IL-18, IL-33, IL-27, TARC, ENA-78, MIP-3α, MIP-1β, IP-10, SCF and ICAM-1 (Fig. 14). In addition, both intermediate cells and cells contained in a cell preparation derived from dental pulp do not or almost do not express IL-21, exotaxin, MIP-1a, MIG, I-TAC and GM-CSF. The expression of IFN-a is not detected in intermediate cells, but trace amounts of IFN-a are expressed in the cells contained in the cell preparation obtained from the dental pulp. In addition, although not shown in the table, both the intermediate cells and the cells contained in the cell preparation obtained from the dental pulp do not or almost do not express IL-2, IL-4, IL-5, IL-9 and IL-13, which are inflammatory cytokines.

(Results: Difference in expression levels of various factors in unstimulated groups between intermediate cells and cells contained in a cell preparation obtained from dental pulp)

Concentration ratios of factors whose expression levels are relatively different between intermediate cells and cells contained in a cell preparation derived from dental pulp among various factors contained in culture supernatants are shown in FIG. The levels of expression of these factors in the cells contained in the cell preparation obtained from the dental pulp are high compared to the intermediate cells. In particular, the expression levels of IL-6, IL-11, HGF, IGFBP-4, TIMP-3 and TIMP-1 in the cells contained in the preparation of cells derived from dental pulp are 1.5 times or more higher than its expression levels. in intermediate cells.

(Results: change in IL-6 expression level as a result of TNF-α and IFN-γ stimulation)

The concentrations of IL-6 contained in the culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. In intermediate cells, the expression level of IL-6 is increased due to both TNF-α and IFN-γ stimulation compared to no stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: Change in IL-11 expression level as a result of TNF-α and IFN-γ stimulation)

The concentrations of IL-11 contained in the culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 17. In intermediate cells, the expression level of IL-11 is increased due to both TNF-α and IFN-γ stimulation compared to no stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: Change in IP-10 expression level as a result of TNF-α and IFN-γ stimulation)

IP-10 concentrations contained in culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 18. In intermediate cells, the expression level of IP-10 is increased due to both TNF-α and IFN-γ stimulation compared to no stimulation. Although the concentration of IP-10 in the absence of stimulation is not displayed in FIG. 18, this is because the concentration of IP-10 is low, and as shown in FIG. 14, IP-10 is expressed in intermediate cells even in the absence of stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: Change in MCP-1 expression level due to TNF-α and IFN-γ stimulation)

The concentrations of MCP-1 contained in culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 19. In intermediate cells, the expression level of MCP-1 is increased due to both TNF-α and IFN-γ stimulation compared to no stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: Change in GM-CSF expression level due to TNF-α and IFN-γ stimulation)

The concentrations of GM-CSF contained in culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 20. In the absence of stimulation, GM-CSF expression is difficult to recognize both in intermediate cells and in cells contained in a cell preparation obtained from dental pulp. In intermediate cells, GM-CSF expression is induced by TNF-α stimulation but is not induced by IFN-γ stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: change in HGF expression level due to TNF-α and IFN-γ stimulation)

The concentrations of HGF contained in culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 21. In intermediate cells, the level of HGF expression decreases due to TNF-α stimulation, but increases due to IFN-γ stimulation compared to no stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

(Results: Change in IL-8 expression level due to TNF-α and IFN-γ stimulation)

The concentrations of IL-8 contained in culture supernatants upon stimulation with TNF-α and IFN-γ will be shown in FIG. 22. In intermediate cells, the expression level of IL-8 increases due to TNF-α stimulation, but does not change with IFN-γ stimulation compared to no stimulation. The same applies to the cells contained in the cell preparation obtained from the dental pulp.

Example 28 Properties (Cell Dividing Ability) of Cells Derived from Dental Pulp

The frozen intermediate cell product obtained in Example 7 and the cell preparation obtained from dental pulp obtained in Example 16 were thawed and centrifuged (1500 rpm, 5 minutes) to pellet the cells. The cells were then suspended in DMEM (10% FBS), plated in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² , and cultured so that they became 90% to 100% confluent. The cells were washed with PBS, trypsin-EDTA was added to them, and each mixture was allowed to stand for 5-10 minutes at 37°C to detach the cells. DMEM medium (10% FBS) was added there to suspend the cells, and the number of living cells was measured. Then, the cells were seeded in a cell culture plate so that they had a density of 5,000 to 15,000 cells/cm ² and cultured so that they became 90-100% confluent. Cell subculture was repeated, and the number of living cells was measured each time subculture was completed to calculate the number of cell divisions. The number of cell divisions was calculated from the number of living cells at the beginning of each subculture and the number of living cells at the end of culture according to the following equation.

Number of cell divisions = log2 (number of live cells at end of culture/number of live cells at start of culture)

(Results)

The cells contained in the frozen intermediate cell article had the ability to undergo at least 14 divisions even after thawing, and the doubling time at that time was in the range of 3 days (in the range of 72 hours). Furthermore, the cells contained in the dental pulp-derived cell preparation had the ability to undergo at least 4 divisions even after thawing, and the doubling time at that time was in the range of 3 days (in the range of 72 hours).

Example 29: Example of using 1 cell preparation derived from dental pulp

The cell preparation derived from dental pulp has been administered to patients with autoimmune diseases such as rheumatoid arthritis and collagenosis by means such as intravenous drip infusion and local injection as medicines to alleviate various symptoms associated with the diseases.

Example 30: Example of using 2 cell preparation derived from dental pulp

The cell preparation obtained from the dental pulp is thawed during use. During use, the cell preparation obtained from the dental pulp is removed from the liquid nitrogen storage tank and thawed by heating in a water bath at a temperature of 36.5°C to 37.5°C. The thawed cell preparation obtained from dental pulp can be administered to humans as pharmaceutical products by means such as intravenous drip infusion or local injection. In the case of intravenous drip infusion, the cell preparation obtained from the dental pulp is transferred to the dialysis bag from the cell cryopreservation container and then administered to the patient via intravenous drip infusion. In the case of local injection, the cell preparation from the dental pulp is transferred into a syringe from a container for cell cryopreservation, and then administered topically to the patient.

Example 31: Example of using a 3 cell preparation derived from dental pulp

Cell preparations obtained from the dental pulp are thawed before use and used in medical institutions. Accordingly, dental pulp-derived cell preparations that have been cryopreserved in the storage facilities of their manufacturers, distributors, etc. are sent in a frozen state in response to requests from medical institutions and delivered to medical institutions. Cells that have been transferred in a frozen state are thawed immediately before being administered to patients, and then administered to patients by intravenous drip infusion or the like in medical facilities.

Industrial Applicability

The present invention is useful, for example, to provide pharmaceutical products that can be administered to humans and that contain cells derived from dental pulp as active ingredients.

Claims (52)

1. A method for obtaining cells derived from the dental pulp, enriched with pluripotent stem cells, including: step (a) hydrolyzing the dental pulp with collagenase and neutral metalloprotease to prepare a dental pulp suspension, step (b) culturing the suspension for proliferation of pluripotent stem cells contained in suspension at 37°C in the presence of 5% CO ₂ in DMEM medium supplemented with fetal bovine serum at a concentration of 20% (v/v) and antibiotics, when replacing the medium every 2-4 days step (c) freezing the proliferated pluripotent stem cells in a state in which the pluripotent stem cells are suspended in the first cryopreservation liquid, step (d) thawing the frozen pluripotent stem cells, step (e) cultivating the defrosted pluripotent stem cells in a state in which the pluripotent stem cells are attached to the surfaces of the carrier particles to proliferate the pluripotent stem cells on the surface of the carrier particles at 37°C in DMEM medium supplemented with fetal bovine serum at a concentration of 10% (vol. /v) when feeding with glucose, step (f) contacting the carrier particles with trypsin to separate the pluripotent stem cells attached to the surfaces of the carrier particles from the carrier particles, and step (g) preparing a suspension of the separated pluripotent stem cells. 2. The production method according to claim 1, characterized in that qualitative tests of pluripotent stem cells are performed in step (c) or (d). 3. The production method according to claim 2, characterized in that qualitative tests are performed on cells fractionated from cells before being suspended in the first cryopreservation liquid, on subcultured cells that were fractionated from cells before being suspended in the first cryopreservation liquid, on pre-frozen cells that have been suspended in the first cryopreservation liquid and fractionated, on cells immediately after thawing that have been fractionated and frozen, and/or on cells obtained by fractionation, freezing, thawing and culture. 4. The production method according to claim 3, characterized in that qualitative tests are performed for one or more of the following qualitative tests a-j: a qualitative test a which checks that the proportion of cells showing a substantially fusiform shape in all cells when observed under an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9% or greater or equal to 99.95%, a qualitative test b, which checks that the cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90% or greater than or equal to 95%, a qualitative test c that checks that cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression profiles, or checks that cells are positive for at least one of CD73 and CD90 and negative for CD34 in expression profiles of cell surface antigen markers, qualitative test d, which checks that cells are negative for CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression profiles, or checks that cells are negative for at least one of CD40, CD80, CD86 and antigen MHC class II in expression profiles of cell surface antigen markers, qualitative test e, which checks that cells remain negative for CD40, CD80, and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression profiles when cells are stimulated with interferon-γ, or checks that at least any of cells are CD40 negative, CD80 negative, CD86 negative, or MHC class II antigen positive in cell surface antigen marker expression profiles when cells are stimulated with interferon-γ, qualitative test f, which checks that the cells express prostaglandin E2 and/or vascular endothelial growth factor (VEGF) and the expression level of prostaglandin E2 is increased by stimulation of TNF-α cells, qualitative test g, which checks that the expression level of aggrecan increases when cells are cultured in a medium containing a substance that induces differentiation into chondrocytes, a qualitative h test that confirms that the amount of calcium accumulated in cells increases when cells are cultured in a medium containing a substance that induces differentiation into osteocytes, a qualitative test i which confirms that the cells have the ability to divide at least 10, 14 or 15 times in the cell culture plate, and a qualitative test j which checks that the average cell doubling time in the cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours or 36 hours during the qualitative test period i. 5. The method of obtaining according to any one of paragraphs. 1-4, further comprising loading the suspension onto a filter membrane to collect cells past step (g). 6. The production method according to claim 5, characterized in that the filter membrane has a pore diameter of 20 to 80 µm. 7. The method of obtaining according to any one of paragraphs. 1-6, characterized in that the dental pulp is obtained from permanent or temporary human teeth. 8. The method of obtaining according to any one of paragraphs. 1-7, characterized in that the collagenase includes collagenase I, collagenase II, or a mixture thereof. 9. The method of obtaining according to any one of paragraphs. 1-7, characterized in that the neutral metalloproteinase includes thermolysin, dispase, or a mixture thereof. 10. The method of obtaining according to any one of paragraphs. 1-9, characterized in that the first cryopreservation fluid used in step (c) comprises Ringer's bicarbonate solution, human serum albumin and DMSO. 11. The method of obtaining according to any one of paragraphs. 1-10, characterized in that the carrier particles used in step (e) are substantially spherical in shape, having a diameter of 80 to 300 µm in the swollen state. 12. The production method according to claim 11, characterized in that the carrier particles are porous carrier particles having pores with a diameter of 3 to 40 microns, which are open on the surface of the carrier particles in a swollen state. 13. The method of obtaining according to any one of paragraphs. 1-12, characterized in that the carrier particles contain gelatin. 14. The method of obtaining according to any one of paragraphs. 1-13, characterized in that the protease used in step (f) comprises a serine protease, a metalloprotease, or a mixture thereof. 15. The method of obtaining according to any one of paragraphs. 1-13, characterized in that the protease used in step (f) includes trypsin. 16. The method of obtaining according to any one of paragraphs. 1-15 further comprising the step (h) of freezing the suspension obtained in step (g) in a state in which the suspension is suspended in the second cryopreservation liquid. 17. The production method according to claim 16, wherein the second cryopreservation fluid used in step (h) comprises Ringer's bicarbonate solution, human serum albumin and DMSO. 18. The method of obtaining according to any one of paragraphs. 1-15, characterized in that qualitative tests of pluripotent stem cells are carried out at stage (g). 19. The production method according to claim 16 or 17, characterized in that qualitative tests of pluripotent stem cells are carried out at stage (g) or (h). 20. The production method according to claim 18 or 19, characterized in that qualitative tests are carried out on cells fractionated from cells before being suspended in the second cryopreservation liquid, on subcultured cells that were fractionated from cells before being suspended in the second cryopreservation liquid, on pre-frozen cells that have been suspended in the second cryopreservation fluid and fractionated, on cells immediately after thawing that have been fractionated and frozen, and/or on cells obtained by fractionation, freezing, thawing and culture. 21. The production method according to claim 18 or 20, characterized in that qualitative tests are performed for one or more of the following qualitative tests a-j: a qualitative test a' which checks that the proportion of the number of cells showing a substantially fusiform shape in all cells when observed under an optical microscope is greater than or equal to 99%, greater than or equal to 99.5%, greater than or equal to 99.9% or greater or equal to 99.95%, a qualitative test b' that checks that the cell viability is greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90% or greater than or equal to 95%, a qualitative test c' that checks that the cells are positive for CD73, CD90, CD105 and CD166 and negative for CD34 and CD45 in cell surface antigen marker expression profiles, or checks that the cells are positive for at least one of CD73 and CD90 and negative for CD34 in expression profiles of cell surface antigen markers, a qualitative test d' that checks that cells are negative for CD40, CD80, CD86 and MHC class II antigen in cell surface antigen marker expression profiles, or checks that cells are negative for at least one of CD40, CD80, CD86 and MHC class II antigen in expression profiles of cell surface antigen markers, a qualitative test e' that checks that cells remain negative for CD40, CD80 and CD86 and become positive for MHC class II antigen in cell surface antigen marker expression profiles when cells are stimulated with interferon-γ, or checks that at least any of cells are CD40 negative, CD80 negative, CD86 negative, or MHC class II antigen positive in cell surface antigen marker expression profiles when cells are stimulated with interferon-γ, qualitative test f, which checks that the cells express prostaglandin E2 and/or vascular endothelial growth factor (VEGF) and the expression level of prostaglandin E2 is increased by stimulation of TNF-α cells, qualitative test g', which checks that the expression level of aggrecan increases when cells are cultured in a medium containing a substance that induces differentiation into chondrocytes, a qualitative test h', which checks that the amount of calcium accumulated in cells increases when cells are cultured in a medium containing a substance that induces differentiation into osteocytes, i’ qualitative test, which checks the ability of cells to divide at least 3, 4 or 5 times in a cell culture plate, and a qualitative test j' that checks that the average cell doubling time in a cell culture plate is 96 hours, 84 hours, 72 hours, 48 hours or 36 hours during the qualitative test period i'. 22. The method of obtaining according to any one of paragraphs. 18-21, characterized in that the CD107b positivity rate in the pluripotent stem cells used in the qualitative tests in step (g) or (h) is higher than the corresponding positivity rate for the pluripotent stem cells used in the qualitative tests in step (c) or (d). 23. The method of obtaining according to any one of paragraphs. 18 - 22, characterized in that the positivity rate of at least one of CD39, CD49a, CD61, CD107a, CD107b and CD143 in pluripotent stem cells used in qualitative tests at stage (g) or (h) is higher than the corresponding indicator positivity in the pluripotent stem cells used in the qualitative tests in step (c) or (d), and wherein the CD146 positivity rate in the pluripotent stem cells used in the qualitative tests in step (g) or (h) is lower than the corresponding positivity rate in the pluripotent stem cells used in the qualitative tests in step (c) or (d). 24. The method of obtaining according to any one of paragraphs. 18-23, characterized in that the level of expression of at least one of IL-6, HGF, IGFBP-4, IL-11, TIMP-3 and TIMP-2 in the used pluripotent stem cells in the qualitative tests at stage (g) or (h) higher than the corresponding level of expression in the pluripotent stem cells used in the qualitative tests in step (c) or (d).

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