JP2002080377A - Method for regenerating in vivo tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for reproducing an in vivo tissue by separating and concentrating a cell for anagenesis by a simple operation and using the separated and concentrated cell for anagenesis. SOLUTION: This method for reproducing an in vivo tissue comprises collecting a cell floating solution containing a cell for anagenesis useful for reproduction of an in vivo tissue and an admixture cell, passing the collected cell floating solution through a cell separation filter which passes at least the admixture cell and catches the cell for anagenesis, introducing a fluid to the cell separation filter and recovering the cell for anagenesis once collected by the cell separation filter and using the recovered cell for anagenesis for the reproduction of an in vivo tissue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for separating tissue regeneration cells from a cell suspension containing tissue regeneration cells and contaminant cells used for regeneration of living tissue, and using the same for regeneration of living tissue. . More specifically, the present invention relates to separation of cells used for repairing and regenerating lesions and / or defects in living tissue using so-called tissue engineering and a method for repairing and regenerating using the cells. The obtained cells can be used in the treatment of various biological tissue lesions and / or defects using the cells for biological tissue regeneration and in basic science fields such as immunology and cell biology.

[0002]

2. Description of the Related Art In recent years, stems and / or progenitor cells of living tissues or organs (hereinafter simply referred to as tissues) have been used.
The tissue is formed in vitro or in vivo to treat tissue lesions and / or defects.
g. Regenerative medicine has attracted a great deal of attention, and research and development has been actively conducted in various countries around the world (for example, “Monthly Tissue Culture Engineering” Vol. 24, No. 4, Special Issue: Tissue Engineering I (1998) April);
5, Special Issue: Tissue Engineering II (May 1998)).

It is apparent that bone marrow and blood (peripheral blood, umbilical cord / placental blood) contain mesenchymal stem / progenitor cells, which are stem / progenitor cells of cells forming bone, cartilage, blood vessels, and the like. (For example, WO95 / 25164, WO97 / 26326, British Patent GB
No. 2301114A). Contaminant cells such as erythrocytes are often mixed in the site where these cells are present, and it is necessary to remove the contaminant cells and concentrate and separate the cells for tissue regeneration. In International Publication WO95 / 25164, Ficoll-Hypaque specific gravity centrifugation is used.
A method for concentrating and separating cells for tissue regeneration from peripheral blood is disclosed. In addition, British published patent GB2301114A
Japanese Patent Application Laid-Open Publication No. H11-139,055 discloses a method for concentrating and separating cells for tissue regeneration from bone marrow using Percoll specific gravity centrifugation.
These methods based on specific gravity centrifugation are methods commonly used at the laboratory level such as immunology, cell biology, and laboratory medicine, but they are complicated and operate in a clean bench. Due to the operation of a completely open system, sterility was difficult, and it was not an acceptable method for clinical practice. In order for tissue engineering to move away from laboratory-level experimental medicine and develop into routine medical practice, it has been desired to simplify the separation / concentration operation and perform treatment in a non-completely open system.

Further, for example, when bone marrow is collected from a patient and used for treatment of a bone defect using bone precursor cells in the bone marrow, there is a problem that a large amount of bone marrow cannot be collected because the patient causes anemia. There are solutions to this problem, such as transfusion with allogeneic blood or autologous blood, but the former cannot rule out the risk of viral infection, and the latter requires additional blood sampling and storage separately from bone marrow collection. And create another problem.

On the other hand, in the field of hematology, hematopoietic stem cell transplantation, which is a regeneration of hematopoietic tissue, ie, bone marrow, has already been established as a normal medical practice. For the enrichment and separation of hematopoietic stem cells used in the same field, see, for example, JP-A-8-10
No. 4643, a filter method characterized by simple operation is used. In addition, with the aim of applying biological regeneration cells other than hematopoietic tissues to the treatment of lesions and / or defects in biological tissues and using them in the field of basic science, the present inventors have proposed a biological tissue regeneration method using a filter method. A method for separating cells for use was developed and a patent application was filed (Japanese Patent Application No. 11-345515). In these methods, although a method for separating and recovering hematopoietic tissue or cells for regenerating living tissue other than hematopoietic tissue could be provided, the separated and recovered cells for regenerating living tissue can be effectively used for living tissue regeneration. Did not.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to separate and concentrate cells for tissue regeneration by a simple operation and a non-completely open system,
It is an object of the present invention to provide a method for regenerating a living tissue using the separated and concentrated tissue regenerating cells.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve such problems. As a result, using the filter used to concentrate and separate hematopoietic stem cells, we made a surprising discovery that it is also possible to concentrate and separate cells for tissue regeneration other than hematopoietic tissues. It has been found that living tissue can be regenerated using cells for regeneration. As a result of further study, the present inventors have found specific conditions for a regeneration method suitable for the regeneration of a living tissue from the separated and concentrated cells for living tissue regeneration, and have completed the present invention.

That is, the present invention provides (1) collecting a cell suspension containing cells for tissue regeneration and contaminating cells used for regenerating living tissue, and regenerating tissue by passing at least the contaminating cells through the collected cell suspension. The cells for use are passed through a cell separation filter to be captured, a fluid is introduced into the cell separation filter to collect the cells for tissue regeneration once captured by the cell separation filter, and the collected cells for tissue regeneration are collected from a living tissue. A method for regenerating a biological tissue including the use of regenerating cells, and (2) collecting, from an individual, a cell suspension containing cells for tissue regeneration and contaminating cells used for regenerating a biological tissue, and the collected cell suspension. At least pass red blood cells through and pass through the cell separation filter to capture tissue regeneration cells, and collect red blood cells that have flowed out of the cell separation filter. Introducing a fluid into the cell separation filter to collect the tissue regeneration cells once captured by the cell separation filter, transfusing the collected red blood cells to the same individual or another individual, and regenerating the collected tissue The present invention relates to a method for regenerating a living tissue, which comprises using cells for regenerating a living tissue.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The living tissue referred to in the present invention refers to connective tissues such as bones, cartilage, muscles and tendons, hematopoietic tissues, blood vessels, nerves, skin, hair, and various organs. The cells for regenerating living tissue referred to in the present invention refer to stem and / or progenitor cells of the above-mentioned tissue used for regenerating the above-mentioned tissue, for example, hematopoietic stem cells and / or hematopoietic progenitor cells, mesenchymal cells Examples include, but are not limited to, lineage progenitor cells, vascular endothelial progenitor cells, neural stem cells, and the like. In addition, regeneration of a living tissue is necessary for a lesion, defect, injury, or the like of the tissue, but is not limited thereto. Contaminating cells are often mixed at the site where these cells are present,
It refers to cells such as erythrocytes that do not have the function of regenerating the tissue of the present invention.

The cell suspension containing these may be any one containing erythrocytes and cells for regenerating living tissue. Specifically, bone marrow fluid, cord blood (including not only blood collected from umbilical cord blood vessels, but also blood collected from placental blood vessels),
Peripheral blood, lymph and those obtained by subjecting them to some treatment such as centrifugation, or those obtained by resuspending cells extracted from various organs or tissues in some liquid. For example, bone marrow fluid, umbilical cord blood, and the like are cell suspensions suitably used in the present invention, including hematopoietic stem cells used for regeneration of hematopoietic tissues and tissue regeneration cells used for regeneration of living tissues other than hematopoietic tissues.

In the present invention, the cell separation filter for passing through the contaminating cells and capturing the cells for regenerating a living tissue is a filter in which a filter medium is filled in a container having a liquid inlet and a liquid outlet. As the filter material, any material can be used as long as it is a nucleated cell trapping material that is generally used, but polyethylene, polypropylene, polystyrene, and acrylic resin are preferable in terms of moldability, sterility, and low cytotoxicity. Synthetic polymers such as nylon, polyester, polycarbonate, polyacrylamide, and polyurethane; natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan, and alginate; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and stainless steel , Titanium, aluminum and the like.

Further, examples of the shape of the filter medium include a woven fabric, a nonwoven fabric, a sponge-like structure, and a membrane. In the case of a nonwoven fabric, when so-called bioligands that specifically bind to specific cells such as antibodies described below are not immobilized on the surface, the fiber diameter is usually 1.0 μm or more and 30 μm or less, preferably 1.0 μm or more. It is 20 μm or less, still more preferably 1.5 μm or more and 10 μm or less. 1.0
If it is less than μm, cells that need to be collected may be firmly captured, and may be difficult to collect. On the other hand, if it exceeds 30 μm, the cells that need to be collected are more likely to pass through without being captured by the fibers. Either case is not preferable because it may lead to a decrease in the recovery rate.

When a sponge-like structure is used, the pore diameter is usually from 2.0 μm to 25 μm, preferably from 3.0 μm to 20 μm, and more preferably from 4.0 μm to 15 μm. 2.0 μm
If it is less than 25 μm, the flowability will be remarkably inferior, and it may be difficult to pass the solution itself.

As the material of the container filled with the filter medium and having a liquid inlet and a liquid outlet, preferable materials in terms of excellent moldability and sterility and low cytotoxicity are exemplified by polyethylene, polypropylene, polystyrene, and the like. Synthetic polymers such as acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, and vinyl chloride; inorganic materials such as hydroxyapatite, glass, alumina, and titania; and metals such as stainless steel, titanium, and aluminum. It is not limited. Examples of the structure of the container include a rectangular parallelepiped, a cube, a columnar shape, an elliptical columnar shape, and the like, but any shape may be used. As for the positions of the liquid inlet and the liquid outlet, the liquid inlet may be any position at which liquid can be introduced into the uppermost layer of the filter medium, and the liquid outlet may be a position at which liquid can be extracted from the lowermost layer of the filter medium. Good.

In the method for separating cells for regenerating living tissue according to the present invention, a cell suspension containing the cells for regenerating living tissue and contaminating cells is passed through the cell separation filter, and then the fluid is introduced into the filter. Although the cells for regenerating living tissue that have been captured as a result of the recovery are collected, rinsing may be performed in order to wash away a small amount of contaminating cells remaining on the filter before introducing the recovery fluid. As the rinsing solution, any liquid can be used as long as it does not adversely affect the cells. Some examples include physiological saline, Dulbecco's phosphate buffer (D-PBS) and Hanks' solution (H
BSS) and a culture medium such as RPMI1640. The rinsing solution may be introduced in the same direction or in the opposite direction as the flow direction of the cell suspension containing the cells for living tissue regeneration and contaminating cells, but the captured cells may leak in the same direction. It is more preferable because it tends to be low.

As a fluid to be introduced into the filter for recovering the captured cells, any fluid may be used as long as it does not adversely affect the cells. Some examples thereof include physiological saline and D-PBS (Dulbecco). Buffer solutions such as phosphate buffer solution, HBSS (Hanks solution), RP
A medium such as MI-1640 can be used. Cell protection, nutritional supplementation, anticoagulation, prevention of freezing damage during cryopreservation, and improved viscosity of these liquids (may be effective for improving the recovery rate)
If necessary for purposes such as, albumin, globulin, glucose, saccharose, trehalose, citrate compound, EDTA, dimethyl sulfoxide, dextran,
Polyvinylpyrrolidone, glycerin, chitin derivatives, hydroxyethyl starch, gelatin and the like may be added. In addition, the fluid referred to here is not limited to a liquid alone,
A mixture of gases that do not adversely affect cells, such as air, argon, and nitrogen, is also included. The direction of introduction of the fluid may be the same as or opposite to the direction of flow of the cell suspension containing cells for regenerating living tissue and contaminating cells, but the reverse direction tends to provide a higher recovery rate. More preferred.

In the method for separating cells for living tissue according to the present invention, the contaminating cells pass through a cell separation filter. The contaminating cells that have passed can be collected and used for any purpose. For example, when the cell suspension is bone marrow obtained from a bone-deficient patient and the cells used for regenerating living tissue are bone precursor cells, and the cells are used to treat a bone defect (bone regeneration), The cells are erythrocytes, and the erythrocytes that have flowed out of this cell separation filter are collected and stored in a blood bag or the like, and can be used as a red blood cell sample for basic science experiments or for the purpose of collecting hemoglobin, which is a source of artificial red blood cells. . In addition, blood can be transfused into a patient as blood for transfusion, and anemia due to bone marrow collection can be prevented, which is preferable.

In the method for regenerating a living tissue of the present invention, a cell suspension containing at least cells for tissue regeneration is collected from an individual. The method of collection is appropriately selected, for example, using bone marrow puncture needles for bone marrow. In the present invention, the cells for living tissue regeneration once captured and collected by the cell separation filter can be transplanted into the above-mentioned individual, and can also be used for transplantation to another individual or regeneration of living tissue outside the living body. The red blood cells that have flowed out of the cell separation filter can be used as a red blood cell sample for basic science experiments or for collecting hemoglobin, which is a raw material of artificial red blood cells. In addition, blood can be transfused into a patient as blood for blood transfusion. Particularly, transfusion into the above-mentioned individual is more preferable because it will prevent anemia in the individual.

The cells for regenerating living tissue obtained by the present invention are as follows:
As is or as necessary, further separation and purification, culture, activation, differentiation induction, amplification, gene transfer, cryopreservation,
WO 97/26326 proposes a composite with a bone replacement material such as hydroxyapatite; Bio
med. Mater. Res. (Appl. Biome
d. ) Vol. 48, 1999, Combination with artificial joints, Proceedings of the 34th Annual Meeting of the Japanese Society for Artificial Organs S
After being subjected to various treatments such as compounding with an artificial blood vessel proposed in 1-1, it is used for treatment of lesions and / or defects of various living tissues and for research in the field of basic science. Specifically, in the present invention, 5% Fet is removed from the cell separation filter.
Al Bovine Serum (FBS) containing Min
im Essential Medium (ME
The cell separation solution for regenerating living tissue recovered in M) was subjected to 5% CO ₂
After culturing for 2 weeks in an atmosphere, Glycerophosp
hate, Vitamin C Phosphate,
After further culturing for 2 weeks in MEM containing 15% FBS to which Dexamethasone was added, in vitro
Bone could be formed in oro. The present invention has found, for the first time, the conditions necessary for regenerating a living tissue from cells for tissue regeneration collected from such a cell separation filter.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Embodiment 1 Cell separation filter Polyester nonwoven fabric with average fiber diameter of 2.3 μm (approximately 6
0g / m ² , bulk height about 0.3mm) 18 sheets and average fiber diameter 1
2 μm polyester non-woven fabric (with a basis weight of about 100 g / m ² ,
16 pieces (bulk height: about 0.47 mm) were piled up and cut into 35 mm squares by a push cutter to obtain a cell separation filter material. The cell separation filter material is placed outside the container (length x width x thickness) 4
A 1 × 41 × 18 mm diagonal line having a liquid outlet and a liquid inlet on the polycarbonate container was filled with a polyester nonwoven fabric having an average fiber diameter of 12 μm on the outlet side to form a cell separation filter. As shown in FIG. 1, a tube having a three-way cock 4 having a spike 2 at the tip and a branch to a cell collection bag 6 on the way was connected to the inlet side of the cell separation filter. In addition, cell separation and collection filter 1
A three-way stopcock 5 was provided on the way to the outlet side, and a tube having a terminal connected to the red blood cell bag 3 was connected to obtain a cell separation / collection apparatus shown in FIG.

2. Raw material cell suspension 3 ml of human bone marrow fluid is collected by an ordinary method, immediately mixed with 3 ml of D-PBS supplemented with 6 units / ml of heparin, and immediately before the cell separation operation, 15% Fetal Bovine Seru.
m (FBS, Gibco Co.) containing Minimal Ess
initialMedium (MEM, manufactured by Sigma) 60
The resulting mixture was diluted with the same to prepare a raw material cell suspension.

3. Cell separation and collection operation 1. Spike 2 of the cell separation and collection device prepared in 2. Was connected to the blood bag containing the raw material cell suspension (hereinafter, blood bag). The three-way stopcock 4 is set in a direction in which only the blood bag and the cell separation filter 1 communicate with each other, and the three-way stopcock 5 is set in a direction in which only the cell separation filter 1 and the red blood cell bag are connected with each other. And
The red blood cells flowing out of the filter were collected in a red blood cell bag. Replace the empty blood bag with a bag containing saline, pass 100 ml of saline through the filter,
A small amount of red blood cells remaining in the filter were washed away, and this washing solution was also collected in a red blood cell bag. Next, 15% for three-way cock 5
A 30 ml syringe (Luer lock port) containing 30 ml of FBS-containing MEM is connected, and the three-way stopcock 5 is set so that only the syringe and the cell separation filter 1 communicate with each other. The three-way stopcock 4 communicates only with the cell separation filter 1 and the cell collection bag 6. In the direction you want. Next, the cells trapped in the cell separation / collection filter 1 (hereinafter, cells for tissue regeneration) were collected in the collection bag 6 by manually pushing the plunger of the syringe.

4. 2. Culture operation After 15 ml of the tissue regeneration cells collected in the above was placed in a T75 tissue culture flask and cultured in a 5% CO ₂ incubator for 2 weeks, adherent cells were separated and recovered by a known method using trypsin.

5. Evaluation The number of red blood cells collected in the red blood cell bag 3 was measured. The red blood cell count was measured using an automatic blood cell counter. Table 1 shows the number of red blood cells in the raw material cell suspension, the number of red blood cells in the red blood cell bag, and the red blood cell recovery rate calculated from these.

[Table 1]

The number of nucleated cells collected in the cell collection bag was measured using an automatic blood cell counter. Table 2 shows the number of nucleated cells in the raw material cell suspension, the number of nucleated cells in the cell collection bag, and the nucleated cell recovery rate calculated from these.
Note that not all nucleated cells are tissue regeneration cells, but tissue regeneration cells are included in the nucleated cells.

[Table 2]

The in vitro bone formation was evaluated by the following method. That is, the collected 5 × 10 ⁴ cells were placed in a culture dish having a diameter of 35 mm, and 10 mM Gl was added.
yserophosphate, 82 μg / ml Vi
tam C Phosphate, 10 nM De
After culturing for 2 weeks in MEM containing 15% FBS to which xamethasone (all in the final concentration) was added, evaluation was performed by alizarin staining and alkaline phosphatase staining.

6. Results It became clear that the cells for tissue regeneration can be separated and collected by this separation operation, and that the obtained cells for tissue regeneration cells have the ability to form bone in vitro as shown in FIG. In FIG. 2, calcification is observed in the portion surrounded by the arrow, and it can be confirmed that bone is formed. Further, red blood cells were collected in the red blood cell bag at a high recovery rate of 90% of the red blood cells in the raw cell suspension.

[0028]

Embodiment 2 Cell Separation Filter A similar operation was performed except that the hydrophilic polymer was coated by the following method in order to increase the liquid permeability of the filter of Example 1. That is, 15 mL of a 1% ethanol solution of a hydroxyethyl methacrylate / dimethylaminoethyl methacrylate copolymer (molar ratio: 97: 3) was passed through the liquid inlet of the cell separation filter through the filter of Example 1 and nitrogen gas was used. After purging excess polymer solution, it was dried with a vacuum dryer at 60 ° C. for 8 hours or more to obtain a cell separation filter. This filter was connected to the same circuit as in Example 1 to obtain a cell separation / recovery device.

2. Material cell suspension The same material cell suspension as in Example 1 was used. 3. Cell separation / collection operation The same cell separation / collection operation as in Example 1 was performed.

4. Culture operation The same culture operation as in Example 1 was performed. 5. Evaluation The bone formation and the collected red blood cells were evaluated in the same manner as in Example 1. Table 3 shows the number of red blood cells in the raw material cell suspension, the number of red blood cells in the red blood cell bag, and the red blood cell recovery rate calculated from these.

[Table 3] 6. Results An osteogenic image similar to that of Example 1 was obtained. In addition, red blood cells were collected in the red blood cell bag at a high recovery rate of 91% of the red blood cells in the raw material cell suspension.

[0031]

As described above, according to the present invention, the cells for tissue regeneration in the raw cell suspension can be separated and concentrated in a simple operation in a non-completely open system while maintaining the function. The cells were found to have tissue regeneration capabilities. At the same time, red blood cells can be collected at a high rate, and it is extremely important that tissue engineering can be abandoned from laboratory-level experimental medicine and contribute to the development of routine medical practice.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a cell separation device for regenerating a biological tissue according to a first embodiment.

FIG. 2 shows in vitro cells isolated in Example 1.
3 is a photograph showing a state of bone formation.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Cell separation filter 2 Spike 3 Red blood cell bag 4 Three-way stopcock 5 Three-way stopcock 6 Cell collection bag 7 Cell injection fluid injection means

Continued on front page F-term (reference) 4B065 AA90X AC20 BB25 BC01 BC21 BC42 BD18 CA44 4C077 BB02 CC01 EE01 KK11 LL02 LL12 NN02 NN18 PP08 PP10 PP12 PP13 4C087 AA01 AA02 AA04 BB34 BB44 BB63 DA03 DA17 Z21

Claims (2)

[Claims] 1. A method for collecting a cell suspension containing cells for tissue regeneration and contaminating cells used for regenerating a living tissue, and for passing the collected cell suspension through at least the contaminating cells to capture the cells for tissue regeneration. Passing through a separation filter, introducing a fluid into the cell separation filter to collect tissue regeneration cells once captured by the cell separation filter, and using the collected tissue regeneration cells for regeneration of living tissue And a method for regenerating a living tissue. 2. Collecting a cell suspension containing cells for tissue regeneration and contaminant cells used for regeneration of a living tissue from an individual, and capturing the cells for living tissue regeneration by passing the collected cell suspension through at least red blood cells. Passing through a cell separation filter to be collected, collecting red blood cells flowing out from the cell separation filter, collecting fluid for tissue regeneration once captured by the cell separation filter by introducing a fluid into the cell separation filter, A method for regenerating a living tissue, comprising: transfusing the collected red blood cells into the same individual or another individual; and using the collected cells for tissue regeneration for regenerating a living tissue.