JP6754459B2 - Method for producing amniotic mesenchymal cell composition, method for cryopreservation, and therapeutic agent - Google Patents

Description

The present invention relates to a method for producing a mesenchymal cell composition by separating mesenchymal stromal cells (MSCs) suitable for cell therapy application from the amniotic membrane with high purity and easily. Furthermore, the present invention relates to a method for producing a mesenchymal cell composition cryopreserved, and a therapeutic agent containing the mesenchymal cell composition.

Mesenchymal stem cells are somatic stem cells that have been pointed out to be present in the bone marrow, and have the ability to differentiate into bone, cartilage, and fat as stem cells. Mesenchymal stem cells have attracted attention as a promising cell source in cell therapy, and have recently been known to be present in fetal appendages such as placenta, umbilical cord, and egg membrane.

Currently, mesenchymal stem cells are attracting attention because they have immunosuppressive ability in addition to differentiation ability. Using bone marrow-derived mesenchymal stem cells, acute graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation and inflammatory Clinical trials for Crohn's disease, an intestinal disease, are underway. The present inventors have proceeded with research aimed at clinical application of mesenchymal stem cells derived from fetal appendages to immune-related diseases. So far, the present inventors have stated that fetal appendage-derived mesenchymal stem cells have the same differentiation potential as bone marrow-derived mesenchymal stem cells (Non-Patent Document 6), and that egg membrane-derived mesenchymal stem cells are rat autoimmune. Improving the pathophysiology of the sexual myocarditis model (Non-Patent Document 7), and improving the survival rate of umbilical cord-derived mesenchymal stem cells in the mouse acute graft-versus-host disease (GVHD) model (Non-Patent Document 8). I have reported. Compared to bone marrow-derived mesenchymal stem cells, fetal appendage-derived mesenchymal stem cells can obtain more mesenchymal stem cells at one time, can be mass-cultured in a short period of time and at low cost, and are collected non-invasively. It is possible and has a high immunosuppressive effect (Non-Patent Document 7). From these facts, MSC containing fetal appendage-derived mesenchymal stem cells can be applied to cell therapy for various immune-related diseases due to its remarkable immunosuppressive effect.

So far, a method for obtaining pluripotent human fetal-derived stem cells from the egg membrane, placenta, and amniotic fluid, which are fetal appendages, has been reported (Patent Document 1). Patent Document 1 describes a method of positioning these stem cells as c-kit (CD117) positive and separating them by flow cytometry. In addition, a method for obtaining stem cells / progenitor cells capable of differentiating into various adult or pediatric cells from the placenta and umbilical cord has also been reported (Patent Document 2). In Patent Document 2, stem cells / progenitor cells having the ability to differentiate into cells constituting various organs and tissues (= having the ability to differentiate more than mesenchymal stem cells) are contained in the placenta and umbilical cord, and the separation thereof. The method is described.

Degrading enzymes such as trypsin, collagenase, and dispase are generally used for cell separation containing stem cells and progenitor cells derived from these fetal appendages (Patent Documents 1 and 2, Non-Patent Documents 1 to 4). Of the fetal appendages, the amniotic membrane is divided into the amniotic membrane located closest to the fetal side in contact with amniotic fluid and the chorion located outside it, and MSCs derived from the amniotic membrane (amniotic membrane and chorion) also use degrading enzymes. The method of separation is common (Non-Patent Documents 1 and 4).

The amniotic membrane, which is part of the egg membrane of the fetal appendage, is divided into an epithelial cell layer in contact with amniotic fluid and an extracellular matrix layer containing MSCs beneath it (Fig. 1). Therefore, when the entire amniotic membrane is treated with trypsin, not only the extracellular matrix layer but also the basement membrane supporting the epithelial cell layer is digested, resulting in a problem that a mixture of epithelial cells and MSC is formed. As a solution to this problem, as in Non-Patent Documents 1 to 3, in order to separate MSCs from the amniotic membrane with high purity, epithelial cells are first removed by degrading enzymes or manually, and the remaining extracellular matrix layer remains. There is a method of recovering the MSC by treating the MSC again with a separating enzyme. However, these methods have problems that epithelial cells cannot be completely removed or the amount of MSCs recovered is reduced.

In addition, cryopreservation is essential for the on-demand use of egg membrane MSCs for cell therapy. At the research level, various cell cryopreservation solutions based on dimethyl sulfoxide (DMSO) are commercially available, and in clinical trials of bone marrow MSC, cryopreservation solutions containing 10% DMSO are used, but cell survival after thawing. There was a problem that the rate dropped.

Patent No. 4330995 Patent No. 3934539 Special Table No. 2010-518906

Am J Obstet Gynecol. 2004; 190 (1): 87-92. Am J Obstet Gynecol. 2006; 194 (3): 664-73. Current Protocols in Stem Cell Biology 1 E.5 J Tissue Eng Regen Med. 2007; 1 (4): 296-305. Cytotherapy. 2006; 8 (4): 315-7. Stem Cells. 2008; 26 (10): 2625-33. J Mol Cell Cardiol. 2012; 53 (3): 420-8. Cytotherapy. 2012; 14 (4): 441-50. BMC Biotechnology 2012; 12:49.

Considering the cell preparation of human amniotic membrane-derived MSCs, it is preferable that the production process is as simple as possible in order to eliminate contamination of bacteria, viruses and the like. Multiple enzyme treatments require enzyme removal by washing, centrifugation, etc., which results in a decrease in MSC recovery efficiency. Further, in the known method, not all epithelial cells are removed by the step of degrading enzyme (Non-Patent Document 3 and Example 3), and many epithelial cells are still adhered to the amniotic membrane.

As for the cell preservation solution, DMSO is cytotoxic, so it is desirable to reduce the concentration as much as possible. There is also a report of a cryopreservation solution in which the DMSO content is reduced by using rat bone marrow-derived MSCs (Non-Patent Document 9).

The present invention has been made in view of the above problems, and is a method for producing a mesenchymal cell composition by simply and aseptically separating high-purity amniotic membrane-derived MSCs with only one enzyme treatment. The challenge is to provide. Another object of the present invention is to provide a method for producing cryopreserved mesenchymal cells, which suppresses cell aggregation and is optimized for MSC transplantation. Another object of the present invention is to provide a cell therapeutic agent containing an amnion-derived MSC produced by the above method.

As a result of diligent studies to solve the above problems, the present inventors treated the amniotic membrane with collagenase and thermolysin and / or dispase, and passed the enzyme-treated amniotic membrane through a mesh to obtain mesenchymal cells. It was found that it can be separated with high purity. Furthermore, the present inventors cryopreserve the mixture containing mesenchymal cells in a solution containing 5 to 10% by mass of dimethyl sulfoxide and 5 to 10% by mass of hydroxylethyl starch or 1 to 5% by mass of dextran. It was found that the cryopreserved mesenchymal cells can be produced in a form optimized for MSC transplantation. Furthermore, the present inventors have demonstrated that the mesenchymal cell composition obtained by the above method is useful as a cell therapeutic agent. The present invention has been completed based on these findings.

That is, according to the present specification, the following invention is provided.
(1) A step of enzymatically treating the amniotic membrane with collagenase and thermolysin and / or dispase; and a step of passing the enzyme-treated amniotic membrane through a mesh:
A method for producing a mesenchymal cell composition, which comprises.
(2) Step of collecting cells that have passed through the mesh; and step of culturing the collected cells:
The method for producing a mesenchymal cell composition according to (1), further comprising.
(3) The step of recovering the cells that have passed through the mesh is a step of recovering the mesenchymal cells by centrifugation after diluting the filtrate with a double amount or more of the medium or the balanced salt solution. The method for producing a mesenchymal cell composition according to 2).
(4) The mesenchyme according to any one of (1) to (3), wherein the collagenase concentration is 50 CDU / ml to 1000 CDU / ml, and the thermolysin and / or dispase concentration is 100 PU / ml to 800 PU / ml. A method for producing a system cell composition.
(5) The method for producing a mesenchymal cell composition according to any one of (1) to (4), wherein the enzyme treatment step is a step of treating at 30 to 40 ° C. for 30 minutes or more.
(6) The mesenchymal cell composition according to any one of (1) to (5), wherein the enzyme treatment step is a step of stirring at 10 rpm / min to 100 rpm / min for 30 minutes or more using a stirrer or a shaker. How to make things.
(7) The method for producing a mesenchymal cell composition according to any one of (1) to (6), wherein the amniotic membrane is obtained by caesarean section.
(8) The method for producing a mesenchymal cell composition according to any one of (1) to (7), wherein the pore size of the mesh is 40 to 200 μm.
(9) The method for producing a mesenchymal cell composition according to any one of (1) to (8), wherein the step of passing the amniotic membrane through the mesh is free fall.

(10) A mesenchymal cell composition having a content of CD324 and CD326-positive epithelial cells of 20% or less, a content of CD90-positive cells of 75% or more, and a viable cell rate of 80% or more.
(11) The mesenchymal cell composition according to (11), which is obtained by the method for producing a mesenchymal cell composition according to any one of (1) to (9).
(12) Mesenchymal cells obtained by culturing the mesenchymal cell composition according to (10) or (11) in a medium containing more than 0.05% by mass and 5% by mass or less of albumin. Culture composition.

(13) Including a step of cryopreserving a mixture containing mesenchymal cells in a solution containing 5 to 10% by mass of dimethyl sulfoxide and 5 to 10% by mass of hydroxylethyl starch or 1 to 5% by mass of dextran. , A method for producing cryopreserved mesenchymal cells.
(14) The method for producing a cryopreserved mesenchymal cell according to (13), wherein the solution is a solution further containing human albumin in an amount of more than 0% by mass and 5% by mass or less.
(15) The mesenchymal cell, claim (10) or (11), wherein the mesenchymal cell is contained in the mesenchymal cell composition produced by the method according to any one of (1) to (9). (13) or (14), wherein the mesenchymal cells contained in the mesenchymal cell composition according to (12) or the mesenchymal cells contained in the mesenchymal cell culture composition according to (12). A method for producing cryopreserved mesenchymal cells.
(16) Administration of mesenchymal cells, which comprises a step of thawing the cryopreserved mesenchymal cells obtained by the method according to any one of (13) to (15) and then diluting the mesenchymal cells with an infusion preparation at least 2-fold. A method for producing a composition for use.

(17) The mesenchymal cell composition according to (10) or (11) and / or the mesenchymal cell culture composition according to (12), and / or produced by the method according to (16). A cell therapeutic agent containing a composition for administration of mesenchymal cells as an active ingredient.
(18) The cell therapeutic agent according to (17), which is an injectable preparation.
(19) The cell therapeutic agent according to (17), which is a preparation for transplantation of a cell mass or a sheet-like structure.
(20) The therapeutic agent according to any one of (17) to (19), which is a therapeutic agent for a disease selected from graft-versus-host disease, inflammatory bowel disease, systemic lupus erythematosus, liver cirrhosis, or radiation enteritis.

According to the present invention, human amniotic membrane-derived MSCs can be separated easily and accurately. Therefore, the present invention can be expected to lead to the promotion of industrial use of MSCs, which have been shown to be effective in the field of regenerative medicine.

It is a tissue diagram of a human amniotic membrane. It is a figure explaining the overview of the amniotic membrane-derived MSC separation method concerning this embodiment. It is a surface antigen marker expression analysis on cells obtained by enzyme treatment of human amniotic membrane when the concentration of collagenase is constant and the concentration of thermolysin is changed. It is a HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the concentration of collagenase is kept constant and the concentration of thermolysin is changed. It is a surface antigen marker expression analysis on cells obtained by enzyme treatment of human amniotic membrane when the concentration of thermolysin was changed without containing collagenase at all. It is an HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the concentration of thermolysin was changed without containing collagenase at all. This is a surface antigen marker expression analysis on cells obtained from enzyme treatment of human amniotic membrane when trypsin was used. It is a HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when trypsin was used. It is an HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the thermolysin concentration was fixed at 250 PU / ml and the collagenase concentration was shaken. It is an HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the thermolysin concentration was fixed at 500 PU / ml and the collagenase concentration was shaken. It is a surface antigen marker expression analysis on cells obtained by enzyme treatment of human amniotic membrane when the concentration of collagenase is constant and the concentration of dispase is changed. It is an HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the collagenase concentration was kept constant and the dispase concentration was changed. This is a surface antigen marker expression analysis on cells obtained by enzyme treatment of human amniotic membrane when the concentration of dispase was changed without containing collagenase at all. It is an HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the concentration of dispase was changed without containing collagenase at all. It is a HE-stained image of the tissue remaining on the filter after the enzyme treatment of the human amniotic membrane when the dispase concentration was fixed at 250 PU / ml and the collagenase concentration was shaken. It is a photograph 24 hours after sowing the amniotic membrane-derived MSC rapidly thawed from various cryopreservation solutions in a plastic well. It is a photograph 48 hours after sowing amniotic membrane-derived MSCs rapidly thawed from various cryopreservation solutions in plastic wells. The therapeutic effect of amniotic membrane-derived MSC transplantation in a mouse acute GVHD model is examined by the rate of change in body weight over time. The therapeutic effect of amniotic membrane-derived MSC transplantation in a rat inflammatory bowel disease model was observed in terms of changes in disease activity over time and the length of the large intestine on day 5 of treatment. The therapeutic effect of amniotic membrane-derived MSC transplantation in a mouse systemic lupus erythematosus model was observed by changes in urinary protein over time. The therapeutic effect of amnion-derived MSC transplantation in a rat liver cirrhosis model is examined by the fibrotic area ratio of the liver. The therapeutic effect of amnion-derived MSC transplantation in a rat radiation enteritis model was examined by the number of PAS-positive goblet cells by PAS staining of the rectum.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiment, 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.

[1] Explanation of terms As used herein, the term "fetal appendage" refers to the egg membrane, placenta, umbilical cord, and amniotic fluid. Further, the "egg membrane" is a fetal sac containing fetal amniotic fluid, which is composed of amniotic membrane, chorioamnionitis and decidua from the inside. Of these, the amniotic membrane and chorion originate from the fetus. The "amniotic membrane" is a transparent thin film with few blood vessels in the innermost layer of the egg membrane, and the inner wall is covered with a single layer of epithelial cells having a secretory function to secrete amniotic fluid.

As used herein, "Mesenchymal stromal cell (MSC)" means a mesenchymal stromal cell (MSC) (meaning a cell capable of differentiating into cells constituting one or more various organs or tissues). Refers to cells that contain the above and meet the definition of pluripotent mesenchymal stromal cells (MSCs) advocated by The International Society for Cellular Therapy (Non-Patent Document 5, see below).

Definition of pluripotent mesenchymal cells
i) Adhesive to plastic under culture conditions in standard medium.
ii) The surface antigens CD105, CD73, and CD90 are positive, and CD45, CD34, CD14, CD11b, CD79alpha, CD19, and HLA-DR are negative.
iii) Can differentiate into bone cells, adipocytes, and chondrocytes under culture conditions.

As used herein, the term "mesenchymal cell composition" means any composition including mesenchymal cells, and is not particularly limited, but for example, mesenchymal cells after treatment of sheep membrane with a degrading enzyme. It means a cell suspension containing the cells.
The term "mesenchymal cell culture composition" as used herein means a cell suspension obtained by culturing the above-mentioned mesenchymal cell composition.
The term "composition for administration of mesenchymal cells" as used herein means any composition prepared in a form suitable for administration using the above-mentioned composition for mesenchymal cell administration, and is not particularly limited. For example, more than twice the amount of an infusion preparation in a mixture containing mesenchymal cells in a solution containing 5-10% by mass of dimethyl sulfoxide and 5-10% by mass of hydroxylethyl starch or 1-5% by mass of dextran. In addition, it means a cell suspension obtained.

[2] Method for producing mesenchymal cell composition The method for producing a mesenchymal cell composition of the present invention is a step of enzyme-treating amniotic membrane with collagenase and thermolysin and / or dispase; and enzyme-treated amniotic membrane. It is a method including a step of passing through a mesh.

In an example of the method for producing a mesenchymal cell composition according to the present invention, human amniotic membrane is treated with an enzyme solution containing collagenase adjusted to an optimum concentration and thermolysin and / or dispase only once, and after the enzyme treatment. The amniotic membrane digestive juice can be passed through the mesh. The epithelial cell layer containing the basement membrane that is not digested by collagenase and thermolysin and / or dispase adjusted to the optimum concentration remains in the mesh, and becomes the extracellular matrix layer that is digested by collagenase and thermolicin and / or dispase adjusted to the optimum concentration. Since the contained MSC passes through the mesh, the MSC can be recovered by collecting the cells that have passed through the mesh. Furthermore, the mesenchymal cell composition may be produced by culturing the collected cells.

FIG. 1 is a tissue diagram of human amniotic membrane. As shown in FIG. 1, the amniotic membrane consists of a superficial epithelial cell layer and an underlying extracellular matrix layer, the latter containing MSC. Amortic epithelial cells, like other epithelial cells, characteristically express epithelial cadherin (E-cadherin: CD324) and epithelial adhesion factor (EpCAM: CD326), whereas MSCs use these epithelial-specific surface antigen markers. It is not expressed and can be easily distinguished by FACS (Fig. 3).

FIG. 2 is a diagram illustrating an outline of the amniotic membrane-derived MSC separation method according to the present embodiment. Physical collection of amniotic membrane from human fetal appendages (A). The amniotic membrane is washed with an isotonic solution such as physiological saline (B), the amniotic membrane is immersed in a solution containing an enzyme containing collagenase at an optimum concentration and thermolysin and / or dispase, and the amniotic membrane is shaken and stirred at an appropriate temperature. (C). Since the epithelial cell layer is not separated into individual cells by enzyme treatment targeting only extracellular matrix and remains in a layered structure, when the obtained sheep membrane digestive juice is passed through a mesh (D), the epithelial cell layer becomes a mesh. The MSC that remains on top and is contained in the extracellular matrix layer can pass through the mesh and be recovered. E is a centrifuge tube, F is a culture dish, and G is a photograph of cultured cells.

An embodiment of the present invention will be described in detail as an example.
1. Aseptically collect human fetal appendages from elective caesarean section cases in the operating room.
2. Aseptically and manually remove the amniotic membrane from the fetal appendages.
3. Transfer the amniotic membrane to a sterilizing container (disposable cup) and wash it several times with an isotonic solution such as physiological saline to remove the attached blood.
4. Roughly cut the amniotic membrane with a scalpel or scissors (this step may be omitted). In addition, the amniotic membrane may be stored in a medium at 2 to 8 ° C. for 24 to 48 hours before use.
5. Immerse the amniotic membrane in a solution containing collagenase adjusted to the optimum concentration and thermolysin and / or dispase, and stir with a constant temperature shaker at 37 ° C. for 90 minutes at 60 rpm.
6. As a result, the epithelial cell layer remains one layer, and the MSCs contained in the extracellular matrix layer float in the enzyme-containing solution.
7. Set the Falcon cell strainer (100 mm mesh) in a sterile tube (Falcon 50 ml tube) and filter the cell-containing solution after amniotic membrane digestion by free fall, so that the epithelial cell layer remains on the mesh and only the MSC meshes. Pass through.
8. After diluting the MSC-containing solution that has passed through the mesh with Hanks equilibrium salt solution, pellet the mesenchymal cells by centrifugation at 400 xg for 5 minutes.
9. Dilute with aMEM supplemented with 10% FBS, sow in a plastic flask and incubate.

According to the present invention, as in the prior art, enzyme treatment is performed a plurality of times, and each treatment is repeated for centrifugation and washing to prevent a decrease in recovery rate and contamination with microorganisms, and also for Ficoll density gradient centrifugation and the like. A large amount of homogeneous MSC can be easily prepared in a short time without the need for purification operation.

The combination of enzymes used to separate the amniotic membrane-derived MSC according to the present invention is collagenase, which digests only collagen, and thermolysin and / or dispase, which is a metal proteinase that cleaves the N-terminal side of non-polar amino acids.
In the present invention, a combination of collagenase, thermolysin and / or dispase is used, but an enzyme (or a combination thereof) that releases MSCs and does not decompose the epithelial cell layer can also be used. The preferred combination of collagenase, thermolysin and / or dispase concentrations can be determined by post-enzymatic microscopic observation or FACS. It is preferable that the concentration condition is such that the epithelial cell layer is not decomposed and the MSC contained in the extracellular matrix layer is released.

The concentration of collagenase is preferably 50 CDU / ml to 1000 CDU / ml, and the concentration of thermolysin and / or dispase is preferably 100 PU / ml to 800 PU / ml.

Although collagenase 300CDU / ml alone treated viable cell count was 0.29 × 10 ⁶ cells, the addition of thermolysin increased concentration-dependently the number of viable cells, and the thermolysin 400PU / ml viable cells 1.99 × 10 ⁶ cells It increased up to about 7 times, and the viable cell rate was 91.7% (Example 1, Table 1).
Similarly, the addition of dispase to collagenase 300 CDU / ml increased the number of viable cells in a concentration-dependent manner, and with dispase 200 PU / ml, the number of viable cells increased to 3.02 × 10 ⁶ and the viable cell rate was 83.7%. (Example 6, Table 8). Therefore, more viable cells can be obtained by mixing not only collagenase but also thermolysin or dispase and simultaneously enzymatically treating them.
For collagenase 300 CDU / ml, the optimal thermolysin concentration was 400 PU / ml. In this case, the CD90-positive MSC content was 83.3% and the CD324-positive epithelial cells were 12.8% (Example 1, Table 2). The optimum dispase concentration added to collagenase 300 CDU / ml was 200 PU / ml, and in this case, the CD90-positive MSC content was 83.3% and the CD324-positive epithelial cells were 12.8% (Example 6, Table 9). ..

When thermolysin and dispase were added alone at a concentration higher than 400 PU / ml, a phenomenon in which epithelial cells were disrupted was observed (Example 2, FIG. 6, Example 7, FIG. 14).

In the case of collagenase 300 CDU / ml and thermolysin 250 PU / ml, the content of CD90-positive MSCs was 93.3%, and the number of CD90-positive MSC cells was 1.43 × 10 ⁶ (Example 4, Table 6). In the case of collagenase 300 CDU / ml and dispase 250 PU / ml, the content of CD90-positive MSCs was 91.5%, and the number of CD90-positive MSC cells was 1.49 × 10 ⁶ (Example 8, Table 11).

For the enzyme treatment, it is preferable to immerse the amniotic membrane washed with physiological saline or the like in an enzyme solution and treat it while stirring with a stirrer or a shaker, but other stirring means may be used, in short, the MSC may be released efficiently. .. This releases the MSC contained in the extracellular matrix layer. The enzyme treatment can be carried out by stirring at 10 rpm / min to 100 rpm / min for 30 minutes or more, preferably using a stirrer or a shaker. The upper limit of the enzyme treatment time is not particularly limited, but it can be generally performed within 6 hours, preferably within 3 hours, for example, within 90 minutes. The enzyme treatment temperature is not particularly limited as long as the object of the present invention can be achieved, but is preferably 30 to 40 ° C, more preferably 30 to 37 ° C.

At this time, it is important to make a combination of concentrations that do not decompose the epithelial cell layer. This is because the MSC content decreases relatively when epithelial cells are mixed.

By filtering the enzyme solution containing the free MSCs through the mesh, only the free cells pass through the mesh, and the undegraded epithelial cell layer cannot pass through the mesh and remains in a mesh shape, facilitating free MSCs. Can be collected in. At this time, the pore size (size) of the mesh is not particularly limited as long as it does not contradict the object of the present invention, but is preferably 40 to 200 μm, more preferably 40 to 150 μm, and further preferably 70 to 150 μm. Particularly preferably, it is 100 to 150 μm. By setting the pore size (size) of the mesh to the above range, it is possible to prevent the cell viability from being lowered by dropping the mesh by free fall without applying pressure.

Nylon mesh is preferably used as the material of the mesh. Tubes containing 40 mm, 70 mm, and 100 mm nylon mesh, such as the Falcon cell strainer, which is commonly used for research, are available. In addition, medical mesh cloths (nylon and polyester) used in hemodialysis and the like can be used. In addition, arterial filters (polyester mesh, 40 mm to 120 mm) used during extracorporeal circulation are also available. Other materials, such as stainless steel mesh (wire mesh), can also be used.

When passing the MSC through the mesh, natural fall (free fall) is preferable. Forced mesh passage such as suction using a pump is possible, but it is desirable to use as weak a pressure as possible to avoid damaging the cells.

The meshed MSCs can be recovered by centrifugation after diluting the filtrate with double or more medium or equilibrium salt buffer. If desired, the recovered cells can be grown by culturing to produce a mesenchymal cell culture composition. The medium is αMEM / M199 or a medium based on them containing albumin of more than 0.05% and 5% or less, and DMEM / F12 / RPMI1640 or a medium based on these has reduced proliferative properties. It is preferably an αMEM medium containing 10% or more bovine / human serum, and is cultured in a plastic dish flask at 5% CO _{2 in} an environment of 37 ° C. As the equilibrium salt buffer solution, a buffer solution such as darbecolinic acid buffer (DPBS), Earl parallel salt (EBSS), Hanks equilibrium salt (HBSS), and phosphate buffer (PBS) can be used.

According to the method of the present invention described above, while the content of CD324 and CD326-positive epithelial cells is 20% or less, the content of CD90-positive cells is 75% or more, and the viable cell rate is 80% or more. A mesenchymal cell composition can be produced, and the mesenchymal cell composition is also within the scope of the present invention.

[3] Method for producing cryopreserved mesenchymal cells The method for producing cryopreserved mesenchymal cells according to the present invention contains 5 to 10% by mass of dimethyl sulfoxide (DMSO) and contains 5 to 5 to 10% by mass of hydroxylethyl starch. The method comprises the step of cryopreserving the mixture containing mesenchymal cells in a solution containing 10% by mass or 1 to 5% by mass of dextran.

As a result of intensive studies, the present inventors have found a decrease in the survival rate of MSCs after thawing, which is considered to be caused by cytotoxicity due to high concentration DMSO (Example). It has been found that reducing the DMSO content in the cryopreservation solution is preferable as a cryopreservation solution for human MSCs for cell transplantation from the viewpoint of suppressing cell death. The MSC cryopreservation solution used in the method of the present invention is characterized in that the DMSO content is reduced and instead hydroxylethyl starch (HES) or dextran (such as Dextran40) is added. The cryopreservation solution may further contain human albumin in an amount of more than 0% by mass and 5% by mass or less. As an example of the cryopreservation solution, a cryopreservation solution having a composition of DMSO 5% by mass, HES 6% by mass, and human albumin 4% by mass can be used.
Using the cryopreservation solution described above and using a program freezer, for example, the temperature is lowered to a temperature between -30 ° C and -50 ° C (for example, -40 ° C) at a freezing rate of -1 to -2 ° C / min. The cryopreserved mesenchymal cells can be produced by further lowering the temperature to a temperature between -80 ° C and -100 ° C (for example, -90 ° C) at a freezing rate of -10 ° C / min. ..
The cryopreserved mesenchymal cells obtained by the above method can be thawed and then diluted 2-fold or more with an infusion preparation to produce a composition for mesenchymal cell administration.

[4] Cell Therapeutic Agents The MSCs (including grown MSCs) prepared above can be used as therapeutic agents for intractable diseases.
That is, according to the present invention, there is provided a cell therapy agent containing the above-mentioned mesenchymal cell composition and / or mesenchymal cell culture composition and / or mesenchymal cell administration composition as an active ingredient. Further, according to the present invention, the above-mentioned mesenchymal cell composition and / or mesenchymal cell culture composition and / or mesenchymal cell administration composition used for cell transplantation therapy is provided. .. Further, according to the present invention, a step of administering to a subject a therapeutically effective amount of the above-mentioned mesenchymal cell composition and / or mesenchymal cell culture composition and / or mesenchymal cell administration composition. A method of transplanting cells into a subject and a method of treating a subject's disease are provided.

The cell therapeutic agent of the present invention, the mesenchymal cell composition and / or the mesenchymal cell culture composition and / or the composition for administration of the mesenchymal cell is described in, for example, graft-versus-host disease (GVHD, Example 10). Inflammatory bowel disease including Crohn's disease (Example 11) and ulcerative colitis, collagen disease including systemic lupus erythematosus (Example 12), liver cirrhosis (Example 13), radiation enteritis (Example 14), atopic skin It can be applied to flames and the like. Inflammation can be suppressed by administering the MSC prepared by the production method of the present invention to the treatment site in an amount whose effect can be measured.

In order to maintain the cell viability, the cryopreserved MSCs need to be used as soon as possible after rapid thawing and dilution with infusion preparations such as physiological saline. This is because DMSO contained in the cryopreservation solution is cytotoxic. The mesenchymal cell administration composition diluted with the above infusion preparation is also used for patients with graft-versus-host disease, inflammatory enteritis including Crohn's disease, collagen disease including systemic lupus erythematosus, liver cirrhosis, radiation enteritis, atopic dermatitis, etc. Can be administered intravenously and treated.

The "infusion preparation" in the present specification is not particularly limited as long as it is a solution used for human treatment, but for example, physiological saline, 5% glucose solution, Ringer's solution, Ringer's lactate, Ringer's acetate solution, No. 1 solution. , No. 2 solution, No. 3 solution, No. 4 solution, etc.

The method for administering the cytotherapeutic agent of the present invention is not particularly limited, but for example, subcutaneous injection, intralymph node injection, intravenous injection, intraperitoneal injection, intrathoracic injection or local direct injection, or local direct transplantation. However, it is not limited to these.

Similar to the bone marrow MSC preparation, the cell therapeutic agent of the present invention can also be used as an injection preparation for the purpose of treating other diseases, or as a transplant preparation having a cell mass or a sheet-like structure.

The dose of the cell therapy agent of the present invention is an amount of cells that, when administered to a subject, can obtain a therapeutic effect on a disease as compared with a subject not administered. The specific dose can be appropriately determined depending on the administration form, administration method, purpose of use, age, body weight, symptom, etc. of the subject, and one example is the number of mesenchymal cells of a human (for example, an adult). ^{105 to 109} pieces / kg body weight are preferred per ^{administration,} 105 to ⁸ / kg body weight being more preferred.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.

(Example 1)
The amniotic membrane was manually separated from the human fetal appendages derived from a pregnant woman in a case of elective caesarean section with informed consent. After washing twice with Hanks equilibrium salt solution (without Ca / Mg), take 1 g of the obtained sheep membrane in a container and purify collagenase (CLSPA, Worthington, standard> 500 CDU / mg) 300 CDU / ml (= < 600 μg / ml) and thermolysine (Wako Pure Chemical, standard> 7000 PU / mg) 0 to 400 PU / ml (= <60 μg / ml) (No.1: 0PU / ml, No.2: 100PU / ml, No.3: Add a total of 5 ml of Hanks equilibrium salt solution (containing Ca / Mg) containing 200 PU / ml and No. 4: 400 PU / ml), and shake and stir at 37 ° C for 90 minutes at 60 rpm with a shaker. It was. A double amount of 10% fetal bovine serum (FBS) added αMEM (Alpha Modification of Minimum Essential Medium Eagle) was added to the obtained mixture, and the mixture was filtered through a nylon net filter (pore size: 100 μm). The tissue remaining on the filter was evaluated by hematoxylin and eosin (HE) staining. The filtrate was centrifuged at 400 xg for 5 minutes, the supernatant was discarded, cells were resuspended in αMEM supplemented with 10% FBS, and the number of cells was measured after staining with trypan blue. The obtained cells were stained with the mesenchymal marker anti-CD90-FITC antibody and the epithelial marker anti-CD324-APC antibody (BD Bioscience) at 4 ° C. for 15 minutes, and then 7-AAD dye was added to remove dead cells. Then, surface antigen marker analysis was performed with a flow cytometer (FACSCanto: BD). The results are shown in Table 1, Table 2, FIG. 3 and FIG.

CDU (= collagen digestion unit): The amount of enzyme that produces 1 μmol of leucine-equivalent amino acid and peptide in 5 hours at 37 ° C and pH 7.5 using collagen as a substrate.
PU (= protease unit): The amount of enzyme that produces 1 μg of tyrosine-equivalent amino acid and peptide per minute at 35 ° C and pH 7.2 using lactic acid casein as a substrate.

As shown in Table 1, the number of trypan blue staining-negative viable cells obtained with collagenase alone (No. 1) was 0.29x10 ⁶ , whereas the number of viable cells increased with the addition of thermolysin, and thermolysin was added. At 400 PU / ml (No. 4), it increased to 1.99x10 ⁶ pieces, about 7 times. On the other hand, there was no significant change in the number of trypan blue-positive dead cells, and a viable cell rate of 80% or more was obtained in all samples.

As shown in FIG. 3, from the results of the flow cytometer, in the collagenase-only (No. 1) sample, only 34.6% of the target CD90-positive MSCs, 8.6% of unnecessary CD324-positive epithelial cells, and others. The number of cells that appeared to be red blood cells was 56.8%. As shown in Table 1, the proportion of CD90-positive MSCs increased with the addition of thermolysin, with thermolysin 400 PU / ml (No. 4) having CD90-positive MSCs of 83.3%, CD324-positive epithelial cells of 12.8%, and others of 3.9%. ..

As shown in Table 2, the number of MSCs obtained from each sample was 0.1x10 ⁶ with collagenase alone (No. 1), but the number of MSCs obtained with the addition of thermolysin increased to 400 PU / ml (No. 4). ) and the 1.66X10 ^6, about 16 times the MSC of No.1 was obtained.
As shown in FIG. 4, when the tissue remaining on the filter after the enzyme treatment was examined by HE staining, the structure of the extracellular matrix layer was maintained with collagenase alone (No. 1), and digestion was insufficient. It was. Digestion of the extracellular matrix layer was observed by adding thermolysin, and it was completely digested at 400 PU / ml (No. 4).

From the results of Example 1, the amniotic membrane cannot be digested by collagenase alone, and by adding thermolysin to collagenase, the amniotic membrane is digested in a concentration-dependent manner, and with 400 PU / ml thermolysin, the extracellular matrix layer containing MSC is completely digested. It turned out that it had been digested.

(Example 2)
Based on Example 1 above, a study was conducted using only thermolysin, excluding collagenase, by the same method.
The results are shown in Tables 3, 5 and 6.

As shown in Table 3, the number of cells per 1 g of human amniotic membrane obtained by digestion of thermolysin alone increased in a thermolysin concentration-dependent manner.
However, as shown in FIG. 5, from the results of the flow cytometer of the cells contained in the enzyme treatment solution containing only thermolysin, the cells obtained at any concentration were almost 100% CD324-positive epithelial cells, and the target CD90. No positive MSC was obtained.
As shown in FIG. 6, in the examination by HE staining of the tissue remaining on the filter after the enzyme treatment, the extracellular matrix layer containing MSC was not digested at all, and the disruption of the epithelial cell layer was the concentration of thermolysin. It existed dependently.

From the results of Example 2, it was found that the desired MSC could not be obtained with thermolysin alone, and that the epithelial cell layer was disrupted when the thermolysin concentration was 800 PU / ml or more.

(Example 3)
Furthermore, a comparative study with the conventional method using trypsin was carried out (Non-Patent Document 3). Take 1 g of human sheep membrane in a container and stir with 5 ml of No. 41) 0.05% trypsin (containing 0.53 mM EDTA) at 37 ° C for 90 minutes 60 rpm with shaking (by shaker), No. 42) 0.05% trypsin (containing 0.53 mM EDTA, Invitrogen). After stirring (with a shaker) at 37 ° C for 90 minutes at 60 rpm in 5 ml, the tissue remaining by filtering with a nylon net filter (pore size: 100 μm) is further refined with collagenase (300 CDU / ml) -containing Hanks equilibrium salt solution (Ca. (Mg-containing) at 37 ° C for 90 minutes 60 rpm with shaking (with a shaker), No. 43) Hanks equilibrium salt solution (containing Ca / Mg) containing purified collagenase (300 CDU / ml) + thermolysin (250 PU / ml) to 5 ml Then, shaking and stirring (with a shaker) at 37 ° C. for 90 minutes was performed. The following was the same as in Example 1. The results are shown in Table 4, Table 5, FIG. 7 and FIG.

As shown in Table 4, the largest number of cells could be obtained from the sample (No. 42) in which the remaining amniotic membrane was further treated with collagenase in addition to the trypsin treatment.
However, as shown in FIG. 7, from the results of the flow cytometer of the cells contained in each enzyme treatment solution, the target CD90-positive MSC of 0.6% cannot be obtained with trypsin alone (No. 41) at all. In the case of two-step treatment (No. 42), which was treated with trypsin and then further treated with collagenase, 32.6% of the target CD90-positive cells were obtained, but 65.6% of unnecessary CD324-positive epithelial cells were also contained. In a single treatment of collagenase + thermolysin (No. 43), CD90-positive MSC was 90.3% and CD324-positive epithelial cells were 8.0%.

As shown in FIG. 8, in the examination by HE staining of the tissue remaining on the filter after the enzyme treatment, in addition to the destruction of the basement membrane of the epithelial cell layer, the epithelial cells became spherical and disjointed only by the 41 trypsin treatment. On the other hand, the structure of the extracellular matrix layer was maintained. After the trypsin treatment, the sheep membrane was completely digested by the collagenase treatment (No. 42), and the batch treatment of collagenase + thermolysin (No. 42) was performed. According to 43), the structure of the epithelial cell layer, including the basement membrane, was maintained even though the extracellular matrix layer was completely digested.

As shown in Table 5, MSC obtained from each sample, trypsin only (No.51) the hardly, if you trypsin after collagenase treatment of (No.52), 5.39 x10 ⁶ cells and numerous give It was found that 3.25 x 10 ⁶ pieces were obtained by batch treatment of collagenase + thermolysin (No.53). On the other hand, regarding unnecessary CD324-positive epithelial cells, 2.25 x 10 ⁶ epithelial cells can be obtained with trypsin alone (No. 51), and 13.07 x 10 ⁶ with trypsin + collagenase batch treatment (No. 52). It was clarified that the number of cells exceeding the required MSC was obtained, and that the number of cells was 0.29 x 10 ^{6 in} the batch treatment of collagenase + thermolysin (No. 53), which was smaller than that of MSC.

From the above, the conventional method of treating with collagenase after trypsin treatment has a merit that a large number of cells can be obtained, but a large amount of epithelial cells are contaminated, and a method such as specific gravity centrifugation is used to obtain high-purity MSC. Disadvantages include the need for an operation to select cells and the complicated operation due to the two steps of collagenase treatment in addition to trypsin treatment. In particular, the latter disadvantage is that trypsin is inactivated by Ca, but collagenase is Ca-requiring, so that simultaneous treatment is not possible.

(Example 4)
Based on Examples 1 to 3 above, the minimum collagenase concentration required for amnion mesenchymal cell separation when the thermolysin concentration was fixed at 250 PU / ml was examined. The results are shown in Table 6 and FIG.

As shown in Table 6, the number of trypan blue-staining-negative living cells obtained with collagenase 300 CDU / ml (No. 63) was 1.53 x 10 ⁶ , whereas the collagenase concentration was 75 CDU / ml, which was 1/4 of that. In the case of (No. 61), the number of living cells decreased to 1.16 x 10 ⁶ . There was no significant change in the number of trypan blue-positive dead cells, and a viable cell rate of 80% or more was obtained in all samples. In addition, from the results by the flow cytometer, 90% or more of the CD90-positive mesenchymal cells were found in all the samples.

As shown in FIG. 9, when the tissue remaining on the filter after the enzyme treatment was examined by HE staining, only the epithelial cell layer was found at collagenase concentrations of 300 and 150 CDU / ml (No. 63 and 62). On the other hand, when the collagenase concentration was 75 CDU / ml (No. 61), an extracellular matrix layer was observed although it was a small part, and digestion was insufficient.
From the results of Example 4, when the thermolysin concentration was fixed at 250 PU / ml, the collagenase concentration was preferably at least 75 CDU / ml, more preferably 150 CDU, in order to sufficiently digest the extracellular matrix layer. I found it necessary to make it more than / ml.

(Example 5)
Furthermore, when the thermolysin concentration was fixed at 500 PU / ml, which was twice that of Example 3, the minimum collagenase concentration required for amnion mesenchymal cell separation was examined. The results are shown in Table 7 and FIG.

As shown in Table 7, the number of trypan blue-stained-negative living cells obtained with collagenase 150 CDU / ml (No. 73) was 2.05x10 ⁶ , whereas the collagenase concentration was 1/4 of that, 37.5 CDU /. In the case of ml (No. 71), the number of viable cells decreased to 0.82x10 ⁶ . There was no significant change in the number of trypan blue-positive dead cells, and a viable cell rate of 80% or more was obtained in all samples. In addition, from the results by the flow cytometer, 90% or more of the CD90-positive mesenchymal cells were found in all the samples.

As shown in FIG. 10, when the tissue remaining on the filter after the enzyme treatment was examined by HE staining, only the epithelial cell layer was found at collagenase concentrations of 150 and 75 CDU / ml (No. 73 and 72). On the other hand, when the collagenase concentration was 37.5 CDU / ml (No. 71), many extracellular matrix layers were observed and digestion was inadequate.
From the results of Example 5, when the thermolysin concentration was fixed at 500 PU / ml, the collagenase concentration was preferably at least 37.5 CDU / ml, more preferably 75 CDU, in order to sufficiently digest the extracellular matrix layer. I found it necessary to make it more than / ml.

(Example 6)
Regarding Example 1 above, instead of thermolysin, dispase, which is a metal proteinase that similarly cleaves the N-terminal side of a non-polar amino acid, was used, and a study was conducted in which collagenase was added thereto.
The amniotic membrane was manually separated from the human fetal appendages derived from pregnant women who gave informed consent. After washing twice with Hanks equilibrium salt solution (without Ca / Mg), take 1 g of the obtained sheep membrane into a container and take collagenase (Brightase-C, Nippi, standard> 200,000 CDU / vial) 300 CDU / ml. And Dispase I (Wako Pure Medicine, Standard 10000 ~ 13000PU / Vial) 0 ~ 400PU / ml (No.1: 0PU / ml, No.2: 100PU / ml, No.3: 200PU / ml, No.4: 400PU A total of 5 ml of Hanks equilibrium salt solution (containing Ca / Mg) containing (4 types of / ml) was added, and shaking was stirred at 37 ° C. for 90 minutes and at 60 rpm with a shaker. A double amount of 10% fetal bovine serum (FBS) added αMEM (Alpha Modification of Minimum Essential Medium Eagle) was added to the obtained mixture, and the mixture was filtered through a nylon net filter (pore size: 100 μm). The tissue remaining on the filter was evaluated by hematoxylin and eosin (HE) staining. The filtrate was centrifuged at 400 xg for 5 minutes, the supernatant was discarded, cells were resuspended in αMEM supplemented with 10% FBS, and the number of cells was measured after staining with trypan blue. The obtained cells were stained with the mesenchymal marker anti-CD90-FITC antibody and the epithelial marker anti-CD324-APC antibody (BD Bioscience) at 4 ° C. for 15 minutes, and then 7-AAD dye was added to remove dead cells. Then, surface antigen marker analysis was performed with a flow cytometer (FACSCanto: BD). The results are shown in Table 8, Table 9, FIG. 11 and FIG.

As shown in Table 8, the number of trypan blue staining-negative viable cells obtained with collagenase alone (No. 81) was 0.42x10 ⁶ , whereas the number of viable cells increased with the addition of dispase, and dispase. At 200 PU / ml (No. 83), it increased to 3.02x10 ⁶ pieces, about 7 times. On the other hand, there was no significant change in the number of trypan blue-positive dead cells, and a viable cell rate of 80% or more was obtained in any sample containing dispase.

As shown in FIG. 11, according to the results by the flow cytometer, the target CD90-positive MSC was 90% or more regardless of whether collagenase alone (No. 81) or dispase was added (No. 82-84). There were less than 10% of all unnecessary CD324-positive epithelial cells. The result of collagenase alone (No. 81) was different from that of collagenase alone (No. 1) in Example 1 (Fig. 3), which was considered to be due to the difference in the manufacturer of collagenase used.

As shown in Table 9, the number of MSCs obtained from each sample was 0.39x10 ⁶ with collagenase alone (No. 81), but the number of MSCs obtained with the addition of dispase increased to 200 PU / ml (No. 83). In), 2.86x10 ⁶ pieces, about 7 times as many MSCs as No.81 were obtained.
As shown in FIG. 12, when the tissue remaining on the filter after the enzyme treatment was examined by HE staining, the structure of the extracellular matrix layer was maintained with collagenase alone (No. 81), and digestion was insufficient. It was. Digestion of the extracellular matrix layer was observed by adding dispase, and it was completely digested at 200 PU / ml (No. 83) and 400 PU / ml (No. 84).

From the results of Example 6, the amniotic membrane digestion was insufficient with collagenase alone, and the amniotic membrane was digested in a concentration-dependent manner by adding dispase to collagenase, and the extracellular matrix containing MSC was used for dispase of 2000 PU / ml or more. The matrix layer was completely digested.

(Example 7)
Based on Example 6 above, a study was conducted using only dispase excluding collagenase by the same method.
The results are shown in Table 10, FIG. 13 and FIG.

As shown in Table 10, the number of cells per 1 g of human amniotic membrane obtained by digestion of dispase alone increased in a thermolysin concentration-dependent manner.
However, as shown in FIG. 13, the results of the flow cytometer of the cells contained in the enzyme-treated solution containing only dispase showed that the target CD90-positive MSCs were very few% at any concentration.
As shown in FIG. 14, in the examination by HE staining of the tissue remaining on the filter after the enzyme treatment, the extracellular matrix layer containing MSC was not digested at all, and the disruption of the epithelial cell layer was the concentration of dispase. It existed dependently.

From the results of Example 7, it was found that the desired MSCs could not be obtained by dispase alone, and that the epithelial cell layer was disrupted when the dispase concentration was 800 PU / ml or more.

(Example 8)
Based on Examples 6 to 7 above, the minimum collagenase concentration required for amnion mesenchymal cell separation when the dispase concentration was fixed at 250 PU / ml was examined. The results are shown in Table 11 and FIG.

As shown in Table 11, the number of trypan blue-staining-negative living cells obtained with collagenase 300 CDU / ml (No. 113) was 1.57x10 ⁶ , whereas the collagenase concentration was 75 CDU / ml, which was 1/4 of that. In the case of (No.113), the number of living cells decreased to 1.34 x 10 ⁶ . There was no significant change in the number of trypan blue-positive dead cells, and a viable cell rate of 80% or more was obtained in all samples. In addition, from the results by the flow cytometer, 90% or more of the CD90-positive mesenchymal cells were found in all the samples.

As shown in FIG. 15, when the tissue remaining on the filter after the enzyme treatment was examined by HE staining, the collagenase concentration was 300 CDU / ml (No. 113), whereas only the epithelial cell layer was found. At collagenase concentrations of 75 and 150 CDU / ml (No. 111 and 112), extracellular matrix layers were observed and were poorly digested.
From the results of Example 8, when the dispase concentration was fixed at 250 PU / ml, the collagenase concentration was at least 75 CDU / ml or more, more preferably 150 CDU / ml or more, still more preferable, in order to sufficiently digest the extracellular matrix layer. Found the need for more than 300 CDU / ml.

(Example 9)
The cells obtained in Example 1 were diluted with a 10% FBS-added αMEM culture solution, seeded on a plastic dish, and amnion-derived MSCs were adherently cultured. After cell detachment by trypsin treatment, trypsin is neutralized with αMEM culture medium containing 10% FBS, the supernatant is discarded after centrifugation, and the obtained cell pellet is resuspended in RPMI1640 to prepare an amniotic membrane-derived MSC suspension. did. The cryopreservation solution was adjusted with respect to the obtained suspension so that the final composition was shown in Table 12.

DMSO: Simga-Aldrich, part number D2650
HES: Made by Nipro, part number HES40
Human albumin (Alb): Made by Benesis, 25% blood donated albumin IV 12.5g / 50ml
Dextran 40: manufactured by Otsuka Pharmaceutical Factory, low molecular weight dextran sugar injection physiological saline: manufactured by Otsuka Pharmaceutical Factory: The above dilution adjustment solution was used.

Cryopreservation solution is adjusted to amnion-derived MSC of 10 ⁶ cells / ml, respectively were transferred to 1ml in cryotubes at program freezer, to -40 ℃ at a freezing rate of -1 to-2 ° C. / min Temperature Was further lowered to −90 ° C. at a rate of −10 ° C./min and stored in an ultra-low temperature freezer at −150 ° C. The next day, the cryotube was immersed in a constant temperature bath at 37 ° C. and rapidly thawed. Table 13 shows the percentage of trypan blue-negative living cells of rapidly thawed cells.

From this result, it was clarified that the number of living cells is small only with DMSO of III, and that the proportion of living cells is increased by adding HES, dextran, and albumin.
Furthermore, 100 μL of the thawed cell suspension was seeded in 4 wells of a 24-well plate, 3 ml of αMEM culture medium containing 10% FBS was added, and after 24 hours and 48 hours after photography, the average number of cells per field of view was average. The value was measured. The results are shown in Table 14, FIG. 16 and FIG.

Similar to the results in Table 13, cell proliferation was slow with DMSO III alone or DMSO + Alb IV, but cell proliferation was promoted by the addition of HES and dextran. In particular, remarkable cell proliferation was observed in I, II, and IV in which the concentration of DMSO was reduced.

(Example 10)
Allogeneic bone marrow transplantation and splenocyte transplantation were performed in mice to develop acute graft-versus-host disease (GVHD), and the therapeutic effect of human amnion-derived MSC transplantation was investigated. After 15 Gy X-ray irradiation of 7-8 week old female B6C3F1 mice, allogeneic BDF1 mouse-derived bone marrow cells 1.0 × 10 ⁷ cells and same splenocytes 3 × 10 ⁷ cells were intravenously administered. I transplanted it. Human amniotic membrane-derived MSCs obtained on the 14th, 17th, 21st, and 15th days of bone marrow cell transplantation by the same method as in Example 1 (under the conditions of collagenase 300 CDU / ml + thermolysin 250 PU / ml). 1 × 10 ⁵ cells) were transplanted intravenously, and the body weight was observed over time. FIG. 18 shows the rate of change in body weight from the day of bone marrow cell transplantation.
From this result, it was clarified that the delay in weight gain associated with acute graft-versus-host disease (GVHD) was improved by MSC transplantation derived from human amniotic membrane.

(Example 11)
Inflammatory bowel disease was caused by oral ingestion of dextran sulfate (DSS) in rats, and the therapeutic effect of MSC transplantation derived from human amniotic membrane was investigated. Administration of 8% DSS in free drinking water was started in 8-week-old male SD rats. The day after the start of DSS administration, human amniotic membrane-derived MSC (1 × 10 ⁶ cells) obtained by the same method as in Example 1 (under the conditions of collagenase 300 CDU / ml + thermolysin 250 PU / ml) was intravenously transplanted. DSS was administered for a total of 5 days.
FIG. 19 shows Disease activity index (DAI): weight loss, stool stiffness, rectal bleeding observed, measured and scored) and relative weight changes. From this result, it was clarified that the pathological condition of enteritis was improved by MSC transplantation derived from human amniotic membrane.
The DAI was scored according to the method in the following literature. Cooper, HS; Murthy, SN; Shah, RS; Sedergran, DJ Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab. Invest. 69: 238-249; 1993.

(Example 12)
Systemic lupus erythematosus was caused by administration of Pristane (2,6,10,14-tetramethyl-pentadecane) in mice, and the therapeutic effect of MSC transplantation derived from human amniotic membrane was investigated. Pristane was intraperitoneally administered in 500 μl to 13-week-old male BALB / c mice. At the same time, human amniotic membrane-derived MSC (1 × 10 ⁵ cells / 10 g) obtained by the same method as in Example 1 (under the conditions of collagenase 300 CDU / ml + thermolysin 250 PU / ml) was administered to the tail vein, and the same number was administered every other week thereafter. MSC derived from human amniotic membrane was administered, and biochemical evaluation was performed after 20 weeks.
FIG. 20 shows the course of urinary protein. From this result, it was clarified that proteinuria associated with systemic lupus erythematosus was improved by MSC transplantation derived from human amniotic membrane.

(Example 13)
In rats, repeated administration of carbon tetrachloride (CCl ₄ ) caused liver cirrhosis, and the therapeutic effect of MSC transplantation derived from human amniotic membrane was investigated. Intraperitoneal administration of 2 ml / kg of CCl ₄ was started twice a week for 6-week-old male SD rats. Human amniotic membrane-derived MSC (1 × 10 ⁶ cells) obtained by the same method as in Example 1 (under the conditions of collagenase 300 CDU / ml + thermolysin 250 PU / ml) 3 weeks after the start of CCl ₄ administration was intravenously administered. After transplantation, CCl ₄ was administered for a total of 7 weeks, and histological evaluation of the liver was performed.
FIG. 21 shows the fibrotic area ratio (collagen fiber positive ratio) obtained from the result of Masson trichrome staining of the liver. From this result, it was clarified that the liver fibrosis associated with cirrhosis was improved by MSC transplantation derived from human amnion.

(Example 14)
Radiation enteritis was caused by irradiation of the rectum in rats, and the therapeutic effect of human amniotic membrane-derived MSCs was investigated. 8-week-old male SD rats were irradiated with 5 Gy / day of radiation to the lower abdomen every day for 5 days. Human amniotic membrane-derived MSCs (1 × 10 ⁶ cells) obtained by the same method as in Example 1 (under the conditions of collagenase 300 CDU / ml + thermolysin 250 PU / ml) were intravenously transplanted on the final irradiation day, and the third A histological evaluation of the rectum was performed one day later.
FIG. 22 shows changes in the number of PAS-positive goblet cells (/ HPF: per strongly magnified visual field) obtained from the results of PAS staining of the rectum. From this result, it was clarified that the decrease of goblet cells associated with radiation enteritis was improved by MSC transplantation derived from human amnion.

1 mesenchymal stem cell 2 epithelial cell layer 3 extracellular matrix layer

Claims (10)

A mixture containing 5 to 10% by mass of dimethyl sulfoxide, 4 to 10% by mass of hydroxyethyl starch , and 5% by mass or less of human albumin and containing a mesenchymal cell composition was cryopreserved and then cryopreserved. Including the step of thawing the mixture and then culturing
The method for producing a mesenchymal cell composition, wherein the mesenchymal cell composition has a content of CD324 and / or CD326-positive epithelial cells of 20% or less and a content of CD90-positive cells of 75% or more. The production method according to claim 1 , wherein the viable cell ratio in the mesenchymal cell composition is 80% or more. The production method according to claim 1 or 2 , wherein the CD90-positive cell is a CD90-positive mesenchymal cell. The production method according to any one of claims 1 to 3 , wherein the mesenchymal cells are derived from amniotic membrane. A method for producing a composition for mesenchymal cell administration, which comprises a step of diluting the mesenchymal cells obtained by the method according to any one of claims 1 to 4 to 2 or more with an infusion preparation. A cell therapeutic agent containing mesenchymal cells as an active ingredient obtained by the method according to any one of claims 1 to 5 . A cell therapeutic agent containing the composition for mesenchymal cell administration obtained by the method according to claim 6 as an active ingredient. The cell therapeutic agent according to claim 6 or 7 , which is an injectable preparation. The cell therapeutic agent according to claim 6 or 7 , which is a preparation for transplantation of a cell mass or a sheet-like structure. The cell therapeutic agent according to any one of claims 7 to 9 , which is a therapeutic agent for a disease selected from graft-versus-host disease, inflammatory bowel disease, systemic lupus erythematosus, liver cirrhosis, or radiation enteritis.