# US 10,441,611 B2 · Method for producing amniotic mesenchymal stromal cell composition, method for cryopreserving the same, and therapeutic agent

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,441,611 B2
- Document ID: US10441611B2
- Status: Granted
- Kind: B2
- Application number: 14/912,662
- Filed: 2014-08-18
- Published: 2016-08-11
- Granted: 2019-10-15
- Earliest claimed: 2013-08-19
- Assignees: National Cerebral and Cardiovascular Center; Foundation for Biomedical Research and Innovation At Kobe; Hyogo College of Medicine; National University Corporation Hokkaido University; Kaneka Corporation
- Applicants: National Cerebral and Cardiovascular Center; Foundation for Biomedical Research and Innovation At Kobe; Hyogo College of Medicine; National University Corporation Hokkaido University; Kaneka Corporation
- Inventors: Kenichi Yamahara; Akihiko Taguchi; Toshihiro Soma; Shunsuke Ohnishi; Akira Kobayashi
- Web page: https://patentgrep.com/patent/US10441611B2

## CPC

- A61K 35/50: Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/50: Materials characterised by their function or physical properties {, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 39/00: General protective or antinoxious agents
- C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly}
- C12N 5/0668: {Mesenchymal stem cells from other natural sources}
- C12N 5/0669: {Bone marrow stromal cells; Whole bone marrow (isolated stem cells from bone marrow C12N5/0647, C12N5/0663)}
- A61L 2300/64: Animal cells
- C12N 2506/03: from non-embryonic pluripotent stem cells
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the solution further comprises from more than 0% to 5% by mass of human albumin.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 3)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

A therapeutic method, comprising:
administering a composition obtained by the method according to claim 6 to a patient in need thereof.

### Claim 8 (depends on 7)

The therapeutic method according to claim 7, wherein the composition is injected to the patient.

### Claim 9 (depends on 7)

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.

### Claim 10 (depends on 1)

A therapeutic method, comprising:
administering the cultured mesenchymal stromal cells obtained by the method according to claim 1 to a patient in need thereof.

### Claim 11 (depends on 10)

The therapeutic method according to claim 10, wherein the cultured mesenchymal stromal cells are injected to the patient.

### Claim 12 (depends on 10)

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.

### Claim 13 (depends on 10)

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.