WO2016088373A1

WO2016088373A1 - Cultured cell sheet for transplantation use, method for producing cultured cell sheet for transplantation use, and method for producing bone tissue for transplantation use

Info

Publication number: WO2016088373A1
Application number: PCT/JP2015/005992
Authority: WO
Inventors: 学赤羽; 隆昌清水
Original assignee: 公立大学法人奈良県立医科大学
Priority date: 2014-12-02
Filing date: 2015-12-02
Publication date: 2016-06-09

Abstract

Provided is a cultured cell sheet for transplantation use, which can be stored in a simple manner and rarely undergoes a rejection reaction caused by transplantation immunity without requiring the use of an immunosuppressing agent. The cultured cell sheet for transplantation use is characterized by being produced by washing a cultured cell sheet, which has been produced by culturing mesenchymal stem cells into a sheet-like form on a plate, with PBS and then subjecting the cultured cell sheet to a freezing treatment while immersing the cultured cell sheet in PBS on the plate. Cells in the cultured cell sheet can be killed through the freezing treatment, and a rejection reaction caused by transplantation immunity rarely occurs without requiring the use of an immunosuppressing agent. The mesenchymal stem cells are, for example, bone marrow mesenchymal stem cells. The freezing treatment is, for example, a rapid freezing treatment with liquid nitrogen.

Description

Cultured cell sheet for transplantation, method for producing cultured cell sheet for transplantation, and method for producing bone tissue for transplantation

The present invention relates to a cultured cell sheet for transplantation, a method for producing a cultured cell sheet for transplantation, and a bone tissue preparation method for transplantation.

<Techniques for promoting bone formation against unsettled fusion (false joints) and osteonecrosis after fracture include those using proteins (such as BMP) and those using cultured cells. Typical techniques using cultured cells are 1) a technique for transplanting cultured bone marrow cells with artificial bone, 2) a technique for transplanting cultured bone marrow cells with a gel material, and 3) a technique for transplanting cultured cell sheets. .

Among them, the technique of transplanting a cultured cell sheet is a method in which stem cells collected from a human body are cultured in a medium in a plate (culture dish), and the obtained sheet-shaped cultured cells are placed in a defect such as a skin due to an injury. This is a technique for repairing a defect (Patent Document 1, Non-Patent Document 1).

When producing cultured cell sheets, transplantation with allogeneic cells (transgenic cells, cells of other people) causes immune rejection, so regenerative treatment using living cells generally uses autologous cells. is there. However, complications occur because the cells are collected from the patient himself, and the cell culture is necessary for about one month. If it is possible to use a mass-produced and stocked cultured cell sheet using allogeneic cells, it can be transplanted immediately when needed.

However, the problem here is rejection due to transplantation immunity. If the antigenicity is reduced and only the bone formation promoting action can be maintained even though it is made from an allogeneic cell, an immunosuppressive agent is unnecessary and it becomes an ideal living body-derived transplant material.

Also, there is a demand for the realization of regenerative medicine that measures the regeneration of bone tissue that has fallen into dysfunction or dysfunction.

There is a technique in which a porous carrier material prepared with a bioabsorbable synthetic polymer such as a copolymer of polylactic acid and glycolic acid or a natural polymer such as gelatin is used as a base material for bone prosthesis (Patent Document 2). 3). However, since these are not derived from living tissue, it is difficult to recreate the same form and function as the original tissue.

Research on tissue engineering that reconstructs bone tissue close to the living body by culturing and organizing its own cells taken out from the living body in vitro (Patent Document 4). However, complications occur because the cells are collected from the patient himself, and the cell culture is necessary for about one month.

Therefore, it is conceivable to store frozen cells obtained from other people in a frozen state and regenerate bone tissue using the other cells.

However, the problem here is rejection due to transplantation immunity. If the antigenicity is reduced and the bone formation promoting action can be maintained even though it is a tissue from an allogeneic cell, an immunosuppressive agent is unnecessary and an ideal living body-derived transplantation material is obtained.

JP 2003-275294 A JP 2006-116212 A JP 2004-105046 A JP 2009-240766 A

The present invention has been made in view of such a problem, and can be easily stocked, and a transplanted cultured cell sheet that is less likely to cause rejection due to transplantation immunity without using an immunosuppressant, and for transplantation It aims at providing the manufacturing method of a cultured cell sheet.

It is another object of the present invention to provide a bone tissue preparation method for transplantation, which is a technique for reconstructing bone tissue close to a living body and hardly causes rejection due to transplantation immunity without using an immunosuppressant.

In the cultured cell sheet for transplantation according to the present invention, a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet shape on a plate is washed with a washing solution, and the cultured cell sheet is immersed in the washing solution on the plate. It is characterized by being frozen.

The method for producing a cultured cell sheet for transplantation according to the present invention includes a step of washing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet shape on a plate with a washing solution, and the cultured cell sheet on the plate. And a step of freezing in a state of being immersed in a cleaning liquid.

The bone tissue preparation method for transplantation according to the present invention includes a placing step of placing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet shape on a plate in an environment where biological tissue material exists, and the living body It is characterized by having an extraction step of taking out a cultured cell sheet in which bone tissue is formed from an environment in which tissue material is present, and a sampling step of collecting the bone tissue formed from the cultured cell sheet.

According to the present invention, it is possible to obtain a cultured cell sheet for transplantation and a method for producing a cultured cell sheet for transplantation that can be easily stocked and are less likely to cause rejection due to transplantation immunity without using an immunosuppressant.

Further, according to the present invention, a bone tissue that can reconstruct a bone tissue close to a living body without using an immunosuppressive agent can be obtained.

It is a figure which shows the freeze-thaw cell sheet which freeze-thaw-processed the fresh cell sheet, (a) is a figure which shows the state by the macroscopic observation of the freeze-thaw cell sheet mounted on the culture dish after a freeze-thaw process. And (b) is a 40 × microscope enlarged view of a freeze-thawed cell sheet placed on the culture dish after freeze-thaw treatment, and (c) is a freeze placed on the culture dish after freeze-thaw treatment. It is a 100 times microscope enlarged view of a thawed cell sheet, (d) is a 40 times microscope enlarged view of the frozen thawed cell sheet extract | collected with the scraper from the culture dish after the freeze thaw process. It is a figure which shows the standard line which produced the standard line based on the number of the living cells which produced the fresh cell sheet | seat in the culture dish from which a bottom area differs, and contained five different sizes of fresh cell sheets. It is a figure which shows the state of new-bone tissue formation when the frozen cell sheet which thawed the frozen cell sheet is inject | poured and transplanted to the back part of a Lewis rat subcutaneously, Of which (a) generate | occur | produced in the back part of Lewis rat. It is a figure which shows the macroscopic findings of induration, (b) is a 20 times microscope enlarged view of the incisional tissue section which generate | occur | produced in the back part of Lewis rat, and (c) is the incision of the induration structure which occurred in the back part of Lewis rat. It is a 100 times microscope enlarged view. It is a figure comparing the state of new bone tissue formation at the femur fracture part of Fisher 344 rats, of which (a) is a figure when a frozen thawed cell sheet obtained by thawing a frozen cell sheet is transplanted, ( b) is a diagram showing a control in the absence of transplantation. It is a photograph of hematoxylin and eosin (HE) staining of the formed bone tissue, of which (a) is a microscopic observation, (b) is a diagram showing a region surrounded by the square of (a) It is. It is the photograph figure by Sirius red dyeing | staining of the formed bone tissue, (a) is an observation figure by a microscope, (b) is a figure which shows the area | region enclosed by the square of (a). It is a photograph figure which shows the result of having transplanted the same type | system | group (autologous) fresh cell sheet | seat and the freeze-thaw cell sheet | seat of the same kind. FIG. 3 is a photograph of the formed bone tissue by V Goldner staining. It is a photograph figure of the produced fresh cell sheet | seat, (a) is a figure which shows the state by macroscopic observation, (b) is the microscope enlarged view expanded by 40 time. It is a figure which shows the bone structure | tissue formed in the fresh cell sheet | seat which carried out the implantation | transplantation transplant or artificial bone covering the subcutaneous part of the recipient rat, and was transplanted subcutaneously, and (a) is a figure which shows the state by macroscopic observation in a Lewis rat ( (B shows formed bone tissue), (b) is a magnified view of a tissue microscope magnified 40 times in Lewis rats, and (c) is a diagram showing a state of macroscopic observation in ACI rats (arrows are (D) is an enlarged view of a tissue microscope magnified 40 times in an ACI rat. (E) is a micrograph of a tissue section of a fresh cell sheet subcutaneously transplanted by covering and combining an artificial bone, magnified 20 times, (f) is a magnified photograph of a part of (e) (100 times photograph), arrow Indicates new bone. It is a figure which shows the mRNA expression derived from the formed bone tissue, (a) is a figure which shows mRNA expression of osteocalcin derived from the bone tissue of Lewis rat, (b) is an alkaline phosphatase derived from the bone tissue of Lewis rat. It is a figure which shows the mRNA expression of (ALP). It is a photographic diagram of the formed bone tissue by HE staining, of which (a) is a microscopic observation, (b) is a partially enlarged view of (a), (c) is a square of (b) It is a figure which shows the area | region enclosed by. It is a photographic view of the formed bone tissue by Sirius red staining, of which (a) is a microscopic observation view, (b) is a partially enlarged view of (a), (c) is (b) of It is a figure which shows the area | region enclosed with the square. FIG. 3 is a photograph of the formed bone tissue by V Goldner staining. FIG. 2 is a photographic view of the formed bone tissue by H-E staining, of which (a) is an observation view by a microscope, and (b) is a view showing a region surrounded by a square in (a). It is the photograph figure by Sirius red dyeing | staining of the formed bone tissue, (a) is an observation figure by a microscope, (b) is a figure which shows the area | region enclosed by the square of (a). It is the photograph figure by V * Goldner dyeing | staining of the formed bone tissue, (a) is an observation figure by a microscope, (b) is a partially enlarged view of (a). It is the photograph figure by von Kossa dyeing | staining of the formed bone tissue, (a) is an observation figure by a microscope, (b) is a partially enlarged view of (a). It is a photograph figure which shows the result of having transplanted the syngeneic (in-house) fresh cell sheet and the same kind of fresh cell sheet.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

The cultured cell sheet for transplantation according to this embodiment is obtained by washing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet form on a plate with a washing solution, and freezing the cultured cell sheet immersed in the washing solution on the plate. It has been processed. In other words, the cell sheet for transplantation according to the present embodiment is a cell sheet containing mesenchymal stem cells, and the cell sheet is included in the frozen state in a frozen state and placed on the plate. Has been.

The cultured cell sheet is a mesenchymal stem cell that is allowed to grow in the presence of a medium until it becomes confluent on the plate, and can be peeled off from the plate as a single sheet. The culture period, which is a period until the plate becomes confluent, varies depending on the density of cells, but is, for example, 1 to 2 weeks. The cultured cell sheet is, for example, a single layer and has a thickness of about 5 to 50 μm. The cultured cell sheet may be placed using only the cultured cell sheet, or may be placed as a combination of the cultured cell sheet and an artificial bone.

In the present specification, a cultured cell sheet that has not been subjected to a freezing treatment may be referred to as a fresh cell sheet. A case where the fresh cell sheet is in a frozen state may be referred to as a frozen cell sheet. A case where the frozen cell sheet is in a thawed state may be referred to as a frozen thawed cell sheet. The cultured cell sheet is a concept including the above-described fresh cell sheet, frozen cell sheet, and freeze-thawed cell sheet.

The plate is not particularly limited as long as it is a container usually used for culturing cells, and examples thereof include a petri dish, a flask, a beaker, a tray, and a slide.

As the medium, a medium that satisfies the conditions for suitably proliferating mesenchymal stem cells is appropriately selected, and examples thereof include MEM, α-MEM, DMEM, IMEM, and RPMI-1640. These media are commercially available and may be used alone or in combination of two or more. Additives such as sodium bicarbonate, sodium pyruvate, L-glutamine, and L-alanyl-L-glutamine may be added to the medium as necessary. The medium may also contain serum for the maintenance of cell growth and / or survival. The origin of serum is not particularly limited, and examples include cattle, horses and humans.

Β-glycerophosphate can be added on the last day of the culture period. Since β-glycerophosphate has an action of promoting calcification by supplying phosphate, the cultured cell sheet can be transplanted in a state where bone formation is promoted to some extent. In addition, since the formation of bone is promoted, the tissue surrounded by the bone-like tissue is protected from attack by immune cells, so that immune rejection is unlikely to occur even in cells derived from other families. Furthermore, when β-glycerophosphate is added from the first day of the culture period, the differentiation of bone is promoted and the cultured cell sheet adheres to the plate, and the workability is reduced when the cultured cell sheet is collected from the plate with a scraper or the like. However, by adding it on the last day of the culture period, workability when collecting the cultured cell sheet from the plate is not lowered. When β-glycerophosphate is added on the last day of the culture period, it is preferably added for 6 to 8 hours, for example.

Mesenchymal stem cells can be easily prepared by collecting cells from human bone marrow, adipose tissue, and other tissues and culturing them by a conventional method, preferably bone marrow mesenchymal stem cells. The cultured cell sheet includes osteoblasts, chondrocytes, fibroblasts, stromal cells, adipocytes, vascular endothelium in which mesenchymal stem cells are differentiated in addition to mesenchymal stem cells grown in an undifferentiated state Cells, vascular endothelial progenitor cells, smooth muscle cells, SP cells and the like may be included.

The washing solution for washing the cultured cell sheet is not particularly limited as long as it can wash the cultured cell sheet without destroying cells and proteins contained in the cultured cell sheet. For example, physiological saline, pure water A culture solution, and preferably a phosphate buffer solution (PBS). The pH of PBS is preferably 6-8. The cultured cell sheet is frozen on the plate while immersed in a washing solution. By the freezing treatment, the cells in the cultured cell sheet are killed, and rejection reaction due to transplantation immunity hardly occurs without using an immunosuppressant. This is considered to be because the antigenicity of the cells contained in the cultured cell sheet after being killed by freezing treatment is reduced.

In the present invention, the stock property of the cultured cell sheet for transplantation is improved by freezing treatment. That is, a mass-produced product by freezing can be stocked for a long period of time, and when necessary, it can be thawed and collected as a frozen-thawed cell sheet and transplanted immediately.

In addition, when the cultured cell sheet is frozen by holding with, for example, tweezers, the shape of the cultured cell sheet becomes like a confetti, and the operability is lowered when applied to a bone damage site or the like. Since the sheet is frozen on the plate, the sheet shape can be maintained during the freezing process, and the operability is excellent when applied to a bone damaged site.

Further, in the present invention, since the cultured cell sheet is frozen in a state of being immersed in a washing solution, when it is thawed from the frozen state when applied to a bone damage site or the like, the washing solution adheres to the frozen and thawed cell sheet. Therefore, the flexibility of the freeze-thawed cell sheet can be secured, and the operability when applied to a bone damage site or the like is excellent.

The freezing treatment is not particularly limited, but is preferably a quick freezing treatment with liquid nitrogen. The quick freezing treatment with liquid nitrogen is a method of freezing without forming ice crystals inside and outside the cells. By rapidly cooling, it quickly passes through the original freezing point and becomes supercooled, so that ice crystals are not formed. It utilizes the phenomenon that water molecules stop moving. It is excellent in that the time required for the freezing process is short and no special equipment is required. Although there is no particular limitation on the use of a cultured cell sheet that has been subjected to rapid freezing treatment with liquid nitrogen at the time of transplantation, it is preferable to slowly thaw it.

Note that the slow freezing method can be used as the freezing treatment. The slow freezing method is a technique that can avoid cell damage (freezing damage) due to freezing by cooling the cultured cell sheet over a long period of time at a slow cooling rate and suppressing the formation of ice crystals in the cells. .

Further, the bone tissue preparation method for transplantation according to the present embodiment includes a placing step of placing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet shape on a plate in an environment where biological tissue material is present, It has the extraction process which takes out the cultured cell sheet in which the bone tissue was formed from the environment where biological tissue material exists, and the extraction process which extracts the bone tissue formed from the cultured cell sheet.

The cultured cell sheet, plate, medium, and mesenchymal stem cells are as described above for the cultured cell sheet for transplantation.

Β-glycerophosphate can be added on the last day of the culture period. Since β-glycerophosphate has an action of supplying phosphoric acid to promote calcification, there is an advantage that bone formation for transplantation is promoted. In addition, as explained in the above-mentioned cultured cell sheet for transplantation, since the formation of bone is promoted, the tissue surrounded by the bone-like tissue is protected from attack by immune cells. Even in cells derived from it, immune rejection is unlikely to occur. Furthermore, as described in the above-mentioned cultured cell sheet for transplantation, the workability when collecting the cultured cell sheet from the plate is not lowered by adding it on the last day of the culture period. When β-glycerophosphate is added on the last day of the culture period, it is preferably added for 6 to 8 hours, for example.

Mesenchymal stem cells are differentiated on the plate in the presence of the medium, and in addition to the mesenchymal stem cells grown in an undifferentiated state on the cultured cell sheet, progenitor cells from which the mesenchymal stem cells have differentiated, Osteoblasts, chondrocytes, fibroblasts, SP cells and the like are included. In the process of differentiation, ALP is expressed early, followed by osteocalcin protein expression specific to bone, followed by production of bone matrix and type I collagen in vivo.

The cultured cell sheet is placed in an environment where biological tissue material exists. Here, the biological tissue is not particularly limited. For example, living cells, various proteins such as collagen released by the living cells, extracellular matrices such as sugar, and capillaries that deliver nutrients and oxygen to living cells. , And various biologically active substances in living organisms, specifically, the back of rodents such as rats and the like.

When cells derived from other families are placed in an environment where biological tissue materials are present as they are, there is a possibility that not only the problem of colonization but also immune rejection occurs, but in the present invention, mesenchymal stem cells are present in the presence of the medium. A cultured cell sheet is produced by growing the plate to a confluent state on the plate, and the cultured cell sheet includes not only mesenchymal stem cells but also differentiated progenitor cells and osteoblasts. Moreover, since an extracellular matrix is also formed, immune rejection is unlikely to occur even in cells derived from other families.

After placing the cultured cell sheet in an environment where the biological tissue material exists, for example, a bone tissue is formed on the cultured cell sheet after a period of 7 to 30 days has passed, so the environment where the biological tissue material exists The cultured cell sheet on which the bone tissue is formed is taken out from the above. Thereafter, a bone tissue for transplantation is obtained by collecting the formed bone tissue from the cultured cell sheet.

The obtained bone tissue can be used for the treatment of bone tumors, congenital diseases or bone / joint defects due to trauma, spinal and joint deformities, rheumatic diseases, and the like.

A. Example 1
In Example 1, a fresh cell sheet was prepared, freeze-thawed, a frozen-thawed cell sheet was prepared, and bone formation was attempted by transplanting to a rat.

1 <Preparation of fresh cell sheet>
1-1 <Initial culture>
Bone marrow cells were collected from the femurs of Fisher344 rats and subjected to initial culture in a culture flask (culture area 75 cm ² ) for 14 days. For cell culture, a 37 ° C., 5% CO ₂ incubator was used. On the 14th day of initial culture, the cells were washed twice with PBS (pH = 7.4) and treated with trypsin / EDTA, and bone marrow mesenchymal stem cells were collected as a cell suspension.

1-2 <Secondary culture>
The collected bone marrow mesenchymal stem cells are seeded at a cell density of 1 × 10 ⁴ cells / cm ² in a culture dish (100 mm diameter Falcon dish) used for normal cell culture, and then subjected to secondary culture. As the basic medium, Dulbecco's modified Eagle medium (DMEM + 15% FBS + antibiotic) containing 15% fetal bovine serum (FBS) and an antibiotic (penicillin / streptomycin) was used.

In the secondary culture, dexamethasone and ascorbic acid were added to the basal medium, and the cells were cultured for about 14 days until the cells became confluent to produce a fresh cell sheet. For cell culture, a 37 ° C., 5% CO ₂ incubator was used.

2 <Cell killing treatment>
After reaching confluence in about 14 days of culture, the culture solution was aspirated, washed twice with 5 ml of PBS, and then rapidly frozen with liquid nitrogen while adhering to the culture dish. At the time of quick freezing, the cryoprotection solution was not used, and PBS of the extent that the fresh cell sheet was completely immersed (about 5 ml) was left in the culture dish. A frozen cell sheet was obtained by freezing with liquid nitrogen.

The thawing treatment of the frozen cell sheet was carried out at room temperature (water bath is acceptable), and after confirming that the cell was completely thawed, it was collected as a frozen and thawed cell sheet with a scraper.

The frozen and thawed cell sheet was observed with the naked eye and a microscope immediately after thawing (before collecting the scraper) and after collecting the scraper.

Microscopic observation (strong enlargement) showed cracks in the state of the frozen and thawed cell sheet, but there was little effect on the shape of the frozen and thawed cell sheet after collection with a scraper, and the transplanted state as a frozen and thawed cell sheet was maintained. (Fig. 1).

3 <Confirmation of cell killing effect>
The effect of cell killing treatment with liquid nitrogen was confirmed using a fresh cell sheet prepared in a culture dish (falcon dish) having a diameter of 60 mm.

When viable cells contained in a frozen cell sheet rapidly frozen together with the culture dish were measured, no viable cells could be confirmed. The number of viable cells was measured using Cell Counting Kit-8 (Dojindo Laboratories). This is a cell count kit using tetrazolium salt WST-8, which generates highly sensitive water-soluble formazan, as a chromogenic substrate. WST-8 is reduced by intracellular dehydrogenase to produce water-soluble formazan. By measuring the absorbance of this formazan at 450 nm, the number of living cells can be measured.

Fresh cell sheets were prepared in culture dishes with different bottom areas, and a standard line was prepared based on the number of viable cells contained in five different sizes of fresh cell sheets (FIG. 2). The absorbance of the frozen cell sheet was measured and found to be less than 0.01 (0.00775). It can be seen from the measurement results of the standard line and the frozen cell sheet that the living cells are not contained by the freezing treatment.

4 <Transplantation test>
Recipients used two types of rats (allogeneic rats) with different histocompatibility antigens in consideration of the degree of immune rejection due to differences in major histocompatibility antigens.

A frozen and thawed cell sheet was prepared using Fisher344 rats as donors, and ACI rats and Lewis rats were used as recipients. In transplantation between Fisher344 rats, ACI rats and Lewis rats, immune rejection occurs due to different histocompatibility antigens. Major mismatch and minor mismatch are classified according to the degree of mismatch of histocompatibility antigen. In the case of Fisher344 rats as donors, ACI rats are major mismatches and Lewis rats are minor mismatches. A major mismatch has a greater immune rejection.

4-1 <Transplantation with artificial bone covered>
Artificial bone (β-TCP: β-tricalcium phosphate) was covered with a frozen and thawed cell sheet made of Fisher344 rat bone marrow cells in a 100 mm diameter culture dish (Falcon dish) and transplanted subcutaneously into the rat.

When 4 weeks after transplantation of the frozen and thawed cell sheet, the rat was sacrificed, and the transplanted artificial bone was removed. As a result, a new bone tissue was formed as a histological image.

The extracted artificial bone was fixed with 10% neutral buffered formalin solution for 2 days, decalcified, and embedded in paraffin to prepare a paraffin block. Thin sections were prepared from the blocks, stained with H-E, and new bone formation was observed with a microscope.

4-2 <Injection transplantation with a needle>
Similarly prepared and processed freeze-thawed cell sheets were injected and implanted with 500 μl of PBS subcutaneously in the back of recipient rats (ACI rats and Lewis rats). After 4 weeks, it was excised and confirmed by histology. As a result, new bone tissue was formed (FIG. 3). (c) is an enlarged view of a part of (b), and the arrow in (c) is the formed bone tissue.

4-3 <Transplant to the femoral pseudo-joint model>
A fracture was surgically made on the femur of Fisher 344 rats using a bone saw, and an experiment was conducted in which a frozen thawed cell sheet was transplanted.

The cultured cell sheet for transplantation was used after killing a syngeneic fresh cell sheet derived from bone marrow cells of Fisher 344 rats. The freeze-thaw method was the same as that described above, and the culture dish was 100 mm in diameter.

Fisher 344 rats are inbred rats, and in the case of transplantation using Fisher 344 rats as donors and other Fisher 344 rats as recipients, the genetic background is the same (histocompatibility antigen matches. Same condition as identical twins) and syngeneic transplantation.

After transplantation, the state of transplantation was observed and evaluated. The bone formation was evaluated with X-rays after transplanting a sheet of frozen-thawed cells into the fracture of the rat femur. Callus formation was observed around the fracture at 4 weeks after transplantation (FIG. 4 (a)). In the figure, a thick arrow indicates a fractured portion, and a thin arrow indicates a newly formed callus. No callus formation was observed in the non-transplanted group (control group) (FIG. 4 (b)).
B. Example 2
In Example 2, unlike Example 1, the production conditions of the fresh cell sheet were changed.

1 <Preparation of fresh cell sheet>
Culture conditions were added during the secondary culture. Fisher 344 rat bone marrow cells were cultured in a culture dish having a diameter of 100 mm, dexamethasone and ascorbic acid were added to the basic medium, and the cells were cultured for about 14 days until the cells became confluent. Only on the last day of culture, after culturing for about 6 hours in a medium supplemented with dexamethasone and ascorbic acid in addition to β-glycerophosphate (βGP) in the basic medium, follow the same method as described above. A freezing treatment by, and a thawing treatment at room temperature were performed, and a frozen and thawed cell sheet was collected with a scraper.

2 <Transplantation test>
An experiment was conducted in which the frozen thawed cell sheet prepared and collected from the bone marrow cells of the Fisher 344 rat was transplanted into the ACI rat as a recipient. Since the donor was derived from Fisher 344 rats and transplanted with ACI rats as recipients, the histocompatibility antigen was a major mismatch, and an experimental model in which rejection occurred if no immunosuppressant was used.

2-1 <Injection transplantation with a needle>
A frozen thawed cell sheet prepared from Fisher 344 rat bone marrow cells was implanted and implanted subcutaneously in the back of recipient rats (ACI). After transplantation, no immunosuppressant was administered, and the tissue image was observed after 2 weeks.

2-2 <Transplant to the femur pseudo-joint model>
A fracture was surgically created in the femur of an ACI rat using a bone saw, and a freeze-thawed cell sheet prepared from bone marrow cells of a Fisher 344 rat was transplanted here (transplantation of the same type of frozen-thawed cell sheet). In addition, fractures were surgically made on the femurs of Fisher 344 rats using a bone saw, and fresh cell sheets made from bone marrow cells of Fisher 344 rats were transplanted thereto (syngeneic fresh cell sheet transplantation). No immunosuppressant was administered after transplantation, and the rats were not restricted in walking or loading. Radiographs were taken at 4, 8, 12, and 16 weeks after transplantation to observe the state of bone fusion.

2-3 <Transplantation with artificial bone covered>
Artificial bone (β-TCP) was covered with a frozen thawed cell sheet prepared from Fisher 344 rat bone marrow cells and transplanted subcutaneously to the back of recipient rats (ACI). After transplantation, no immunosuppressant was administered, and the tissue image was observed after 2 weeks.

3 <Observation of transplantation results>
3-1 <Observation of the result of injection transplantation with a needle>
The tissue image of the specimen removed 2 weeks later was observed. Observation was performed by HE staining and Sirius red staining.

FIG. 5 is a photographic view of the formed bone tissue by H-E staining, of which (a) is an observation view by a microscope, and (b) is a view showing a region surrounded by a square in (a). As shown by the arrows in FIG. 5 (b), the formation of bone tissue surrounded by soft tissue was observed. The nucleus of the cells present in the bone tissue could also be observed.

FIG. 6 is a photograph of the formed bone tissue by Sirius red staining, in which (a) is an observation view by a microscope, and (b) is a diagram showing a region surrounded by a square in (a). Sirius red staining stains type I collagen and type III collagen fibers on tissue sections. As indicated by arrows in FIG. 6 (b), even in Sirius red staining using continuous sections of tissue specimens, the sites where bone tissue was observed by H-E staining were positive for Sirius red staining.

3-2 <Observation of the result of transplantation to the femoral pseudo-joint model>
FIG. 7 is a photograph showing the results of syngeneic fresh cell sheet transplantation and the same type of freeze-thaw cell sheet transplantation. The arrow indicates the fracture part. The progress of bone fusion was observed with X-rays. In the group transplanted with the same type of freeze-thawed cell sheet, callus formation was observed at 4 weeks after transplantation, further bone fusion progressed at 8 weeks, and almost complete bone fusion at 12 weeks. The bone fusion rate at 16 weeks was 50%. In the group transplanted with syngeneic fresh cell sheets, callus formation was observed at 4 weeks, bone fusion was completed at 8 weeks, and the bone fusion rate at 16 weeks was 83%.

3-3 <Observation of transplantation result with artificial bone covered>
The tissue image of the specimen removed 2 weeks later was observed. Observation was performed by V Goldner staining.

FIG. 8 is a photograph of the formed bone tissue by V Goldner staining. V Goldner staining stains calcified and uncalcified bones in different colors. As indicated by arrows in FIG. 8, the presence of calcified bone (yellowish green part) could be observed.
C. Example 3
In Example 3, a fresh cell sheet was prepared and transplanted into a rat, and bone formation for transplantation was attempted.

1 <Preparation of fresh cell sheet>
1-1 <Initial culture>
Bone marrow cells were collected from the femurs of Fisher344 rats and subjected to initial culture in a culture flask (culture area 75 cm ² ) for 14 days. For cell culture, a 37 ° C., 5% CO ₂ incubator was used. On the 14th day of initial culture, the cells were washed twice with phosphate buffer (PBS) and then treated with trypsin / EDTA, and bone marrow mesenchymal stem cells were collected as a cell suspension.

1-2 <Secondary culture>
The harvested bone marrow mesenchymal stem cells are seeded at a cell density of 1 × 10 ⁴ cells / cm ² in a culture dish (Falcon dish) used for normal cell culture, and subjected to secondary culture. As the basic medium, Dulbecco's modified Eagle medium (DMEM + 15% FBS + antibiotic) containing 15% fetal bovine serum (FBS) and an antibiotic (penicillin / streptomycin) was used.

At the time of secondary culture, dexamethasone and ascorbic acid were added to the basic medium, and the cells were cultured for about 14 days until the cells became confluent to prepare a fresh cell sheet. For cell culture, a 37 ° C., 5% CO ₂ incubator was used.

After reaching confluence in about 14 days of culture, the culture medium was aspirated, washed twice with PBS, and fresh cell sheets adhering to the culture dish were collected with a scraper. Although it was mechanically collected with a scraper, the shape as a cultured cell sheet was maintained and it was in a transplantable state (FIG. 9).

2 <Mounting test>
Fresh cell sheets made from Fisher344 rat bone marrow cells in a 100 mm diameter culture dish (Falcon dish) were subcutaneously applied to the back of recipient rats (ACI and Lewis rats) with 500 μl of PBS using an injection needle and syringe. Injection transplanted. In addition, since the fresh cell sheet can be transplanted in a form covering artificial bone (β-TCP: β-tricalcium phosphate), the bone formation was confirmed by the placement test in the ACI rat. The skin on the back of the recipient rat was incised and a fresh cell sheet covering the artificial bone was implanted subcutaneously, and then skin suture was performed. Recipients used allogeneic rats considering the degree of immune rejection due to differences in major histocompatibility antigens.

Fresh cell sheets were prepared from Fisher 344 rats (donor), and ACI rats and Lewis rats were used as recipients. In combination with Fisher344 rats, ACI rats have major mismatches and Lewis rats have minor mismatches, and the former has greater immune rejection.

3 <Hard tissue formation and bone tissue collection>
When the injection site was confirmed after 4 weeks, the formation of a hard tissue mass was observed. A site where a tumor was observed was removed and collected, and a tissue section was prepared by HE staining. As a result, a new bone tissue was formed (FIGS. 10A, 10B, 10C, and 10D). A fresh cell sheet covering the artificial bone was excised and collected 4 weeks after subcutaneous. HE staining was performed after the decalcification treatment, and tissue sections were prepared. As a result, new bone tissue was formed (FIGS. 10E and 10F).

4 <Bone formation marker>
RNA was extracted from the extracted hard tissue and the expression of osteocalcin and alkaline phosphatase (ALP) mRNA, which are bone formation markers, was confirmed by real-time PCR, and the expression was confirmed (FIG. 11).
D. Example 4
In Example 4, unlike Example 3, the production conditions of the fresh cell sheet were changed.

1 <Preparation of fresh cell sheet>
Culture conditions were added during the secondary culture. Fisher 344 rat bone marrow cells were cultured in a culture dish having a diameter of 100 mm, dexamethasone and ascorbic acid were added to the basic medium, and the cells were cultured for about 14 days until the cells became confluent. Only on the last day of culture, after culturing for about 6 hours in a medium containing β-glycerophosphate (βGP) in addition to dexamethasone and ascorbic acid in the basic medium, the medium is aspirated, washed with PBS, and then scraped. It was collected as a fresh cell sheet.

2 <Mounting test>
An experiment was conducted in which a fresh cell sheet prepared and collected from the bone marrow cells of the Fisher 344 rat was transplanted into ACI rats as recipients. Since the donor was derived from Fisher 344 rats and transplanted with ACI rats as recipients, the histocompatibility antigen was a major mismatch, and an experimental model in which rejection occurred if no immunosuppressant was used.

2-1 <Transplantation with artificial bone covered>
An artificial bone (β-TCP) was covered with a fresh cell sheet prepared from bone marrow cells of Fisher 344 rats, and transplanted subcutaneously to the back of recipient rats (ACI). After transplantation, no immunosuppressant was administered, and the tissue image was observed after 2 weeks.

2-2 <Transplantation with a needle>
Fresh cell sheets made from Fisher 344 rat bone marrow cells were injected and implanted subcutaneously in the back of recipient rats (ACI). After transplantation, no immunosuppressant was administered, and the tissue image was observed after 2 weeks.

2-3 <Transplant to the femur pseudo-joint model>
A fracture was surgically created in the femur of an ACI rat using a bone saw, and a fresh cell sheet made of bone marrow cells of a Fisher 344 rat was transplanted here (transgenic fresh cell sheet transplantation). In addition, fractures were surgically made on the femurs of Fisher 344 rats using a bone saw, and fresh cell sheets made from bone marrow cells of Fisher 344 rats were transplanted thereto (syngeneic fresh cell sheet transplantation). No immunosuppressant was administered after transplantation, and the rats were not restricted in walking or loading. Radiographs were taken at 4, 8, 12 and 16 weeks after transplantation to observe the state of bone fusion.

3 <Observation of transplantation results>
3-1 <Observation of transplantation result with artificial bone covered>
The tissue image of the specimen removed 2 weeks later was observed. Observation was performed by HE staining, Sirius red staining, and V Goldner staining.

FIG. 12 is a photograph of the formed bone tissue by HE staining, of which (a) is a microscopic observation, (b) is a partially enlarged view of (a), and (c) is (b) ) Is a diagram showing a region surrounded by a square. As shown by arrows in FIG. 12 (c), formation of bone tissue could be observed on the surface of the artificial bone and in the pores.

FIG. 13 is a photograph of the formed bone tissue by Sirius red staining, of which (a) is an observation view by a microscope, (b) is a partially enlarged view of (a), and (c) is ( It is a figure which shows the area | region enclosed by the square of b). Sirius red staining stains type I collagen and type III collagen fibers on tissue sections. As indicated by arrows in FIG. 13 (c), Sirius red staining was also positive, and formation of bone tissue could be observed around the artificial bone.

FIG. 14 is a photograph of the formed bone tissue by V による Goldner staining. V Goldner staining stains calcified and uncalcified bones in different colors. As shown by the arrows in FIG. 14, the presence of calcified bone (yellowish green part) could be observed.

3-2 <Observation of the result of injection transplantation with an injection needle>
The tissue image of the specimen removed 2 weeks later was observed. Observation was performed by HE staining, Sirius red staining, V Goldner staining, and von Kossa staining.

FIG. 15 is a photographic view of the formed bone tissue by H-E staining, of which (a) is an observation view by a microscope, and (b) is a view showing a region surrounded by a square in (a). As shown by the arrows in FIG. 15 (b), formation of bone tissue surrounded by soft tissue was observed. The nucleus of the cells present in the bone tissue could also be observed.

FIG. 16 is a photograph of the formed bone tissue by Sirius red staining, in which (a) is an observation view by a microscope, and (b) is a diagram showing a region surrounded by a square in (a). As indicated by arrows in FIG. 16 (b), even in Sirius red staining using continuous sections of the tissue specimen, the site where bone tissue was observed by H-E staining was positive for Sirius red staining.

FIG. 17 is a photograph of the formed bone tissue by V による Goldner staining, of which (a) is an observation view with a microscope, and (b) is a partially enlarged view of (a). As shown by the arrows in FIG. 17 (b), the presence of calcified bone could be observed.

FIG. 18 is a photograph of the formed bone tissue by von Kossa staining, in which (a) is an observation view by a microscope, and (b) is a partially enlarged view of (a). In von Kossa staining, only calcium phosphate and calcium carbonate in the section are stained black. As shown by the arrow in FIG. 18 (b), it was observed that the deposited calcium salt was dyed black.

3-3 <Observation of the result of transplantation to the femoral pseudo-joint model>
FIG. 19 is a photograph showing the results of transplanting a syngeneic fresh cell sheet and the same type of fresh cell sheet. The arrow indicates the fracture part. The progress of bone fusion was observed with X-rays. In the group transplanted with fresh cell sheets of the same type, callus formation was observed 4 weeks after transplantation, further bone fusion progressed at 8 weeks, and almost complete bone fusion at 12 weeks. The bone fusion rate at 16 weeks was 50%. In the group transplanted with syngeneic fresh cell sheets, callus formation was observed at 4 weeks, bone fusion was completed at 8 weeks, and the bone fusion rate at 16 weeks was 83%. It was confirmed that a bone tissue for transplantation can be sufficiently obtained by collecting a bone tissue formed from a cultured cell sheet even if it is a fresh cell sheet of the same type as well as a fresh cell sheet of the same type.

Can be used as a bone formation treatment material. It can also be used to treat bone tumors, congenital diseases or bone / joint defects due to trauma, spinal cord and joint deformities, or rheumatic diseases.

Claims

For transplantation, wherein a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet form on a plate is washed with a washing solution, and the cultured cell sheet is frozen in a state immersed in the washing solution on the plate. Cultured cell sheet.
The cultured cell sheet for transplantation according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells.
3. The cultured cell sheet for transplantation according to claim 1 or 2, wherein the freezing treatment is a quick freezing treatment with liquid nitrogen.
The cultured cell sheet cultured in a sheet form on the plate is cultured in a medium added with β-glycerophosphoric acid on the last day of the culture period, according to any one of claims 1 to 3. The cultured cell sheet for transplantation as described.
A step of washing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet form on a plate with a washing solution, and a step of freezing the cultured cell sheet in a state immersed in the washing solution on the plate. A method for producing a cultured cell sheet for transplantation, characterized by the following.
The method for producing a cultured cell sheet for transplantation according to claim 5, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells.
The method for producing a cultured cell sheet for transplantation according to claim 5 or 6, wherein the freezing treatment is a quick freezing treatment with liquid nitrogen.
The cultured cell sheet cultured in a sheet form on the plate is cultured in a medium added with β-glycerophosphoric acid on the last day of the culture period, according to any one of claims 5 to 7. The manufacturing method of the cultured cell sheet for transplant of description.
Placing a cultured cell sheet obtained by culturing mesenchymal stem cells in a sheet on a plate in an environment where biological tissue material exists; and
An extraction step of removing the cultured cell sheet in which bone tissue is formed from the environment in which the biological tissue material exists;
And a sampling step for collecting the bone tissue formed from the cultured cell sheet.
The bone tissue preparation method for transplantation according to claim 9, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells.
The mesenchymal stem cell is derived from another family,
The bone tissue preparation method for transplantation according to claim 9 or 10, wherein, in the above-described placing step, the cultured cell sheet is placed in an environment in which a biological tissue material exists without using an immunosuppressive agent.
The bone tissue preparation method for transplantation according to any one of claims 9 to 11, wherein the environment in which the biological tissue material exists is subcutaneous in the back of a rodent.
The bone tissue preparation method for transplantation according to any one of claims 9 to 12, wherein the cultured cell sheet is placed in an environment in which a biological tissue material exists by injection transplantation together with PBS.
14. The bone tissue is formed on the cultured cell sheet after the cultured cell sheet is placed in an environment where the biological tissue material is present and after a period of 1 to 2 months has passed. A bone tissue preparation method for transplantation according to any one of the above.
The cultured cell sheet cultured in a sheet form on the plate is cultured in a medium added with β-glycerophosphoric acid on the last day of the culture period, according to any one of claims 9 to 14. The bone tissue preparation method for transplant as described.