Method for producing amniotic mesenchymal stromal cell composition, method for cryopreserving the same, and therapeutic agent
An object of the present invention is to provide a method for producing a mesenchymal stromal cell composition, comprising conveniently and aseptically separating high-purity amnion-derived MSCs by performing enzyme treatment only once. According to the present invention, the following are provided: a method for producing a mesenchymal stromal cell composition, comprising: performing enzyme treatment of an amnion with collagenase and thermolysin and/or dispase; and filtering the enzyme-treated amnion through a mesh; a method for producing a cryopreserved mesenchymal stromal cell composition; and a therapeutic agent comprising as an active ingredient the mesenchymal stromal cell composition for a disease selected from graft-versus-host disease, inflammatory bowel disease, systemic lupus erythematosus, liver cirrhosis, or radiation enteritis.
1. A method for producing cultured mesenchymal stromal cells, the method comprising:
preparing a mixture of mesenchymal stromal cells and a solution, wherein the mesenchymal stromal cells are mesenchymal stromal stern cells contained in a mesenchymal stromal cell composition;
cryopreserving the mixture;
thawing the cryopreserved mixture; and
culturing the thawed mixture, thereby obtaining the cultured mesenchymal stromal cells,
wherein the mesenchymal stromal cell composition comprises 20% or less of CD324-positive epithelial cells and 75% or more of CD90-positive cells,
wherein the solution comprises from 5% to 10% by mass of dimethylsulfoxide, and from 5% to 10% by mass of hydroxyethyl starch or from 1% to 5% by mass of dextran.
2. The method according to claim 1 , wherein the solution further comprises from more than 0% to 5% by mass of human albumin.
3. The method according to claim 1 , wherein the mesenchymal stromal cell composition is obtained by a process comprising treating an amnion with an enzyme solution comprising collagenase and at least one of thermolysin and dispase.
4. The method according to claim 3 , wherein the enzyme solution comprises from 50 CDU/ml to 1000 CDU/ml of the collagenase and from 100 PU/ml to 800 PU/ml of the total of the at least one of thermolysin and dispase.
5. The method according to claim 1 , further comprising culturing the mesenchymal stromal cells before preparing the mixture of the mesenchymal stromal cells and the solution.
6. A method for producing a composition, comprising:
diluting cultured mesenchymal stromal cells obtained by the method according to claim 1 two-fold or more with an infusion preparation.
7. A therapeutic method, comprising:
administering a composition obtained by the method according to claim 6 to a patient in need thereof.
8. The therapeutic method according to claim 7 , wherein the composition is injected to the patient.
9. The therapeutic method according to claim 7 , wherein the therapeutic method is a method for treating at least one disease selected from the group consisting of a graft-versus-host disease, an inflammatory bowel disease, systemic lupus erythematosus, liver cirrhosis, and radiation enteritis.
10. A therapeutic method, comprising:
administering the cultured mesenchymal stromal cells obtained by the method according to claim 1 to a patient in need thereof.
11. The therapeutic method according to claim 10 , wherein the cultured mesenchymal stromal cells are injected to the patient.
12. The therapeutic method according to claim 10 , wherein the cultured mesenchymal stromal cells are in a preparation for transplantation having a cell aggregate or sheet-like structure.
13. The therapeutic method according to claim 10 , wherein the therapeutic method is a method for treating at least one disease selected from the group consisting of a graft-versus-host disease, an inflammatory bowel disease, systemic lupus erythematosus, liver cirrhosis, and radiation enteritis.