# US 8,465,733 B2 · Pharmaceutical composition containing human mesenchymal stem cell

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- Number: US 8,465,733 B2
- Document ID: US8465733B2
- Status: Granted
- Kind: B2
- Application number: 12/740,758
- Filed: 2008-10-27
- Published: 2011-11-10
- Granted: 2013-06-18
- Earliest claimed: 2007-11-02
- Assignees: JCR Pharmaceuticals Co., Ltd.
- Applicants: Hiroyuki Shirono; Youngjin Byun; Shoichiro Kamei; Kiwamu Imagawa
- Inventors: Hiroyuki Shirono; Youngjin Byun; Shoichiro Kamei; Kiwamu Imagawa
- Web page: https://patentgrep.com/patent/US8465733B2

## CPC

- A61K 9/10: Dispersions; Emulsions
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 35/48: Reproductive organs
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- 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 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 2500/60: Buffer, e.g. pH regulation, osmotic pressure
- C12N 2501/70: Enzymes
- 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)
- 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

Disclosed are a method for producing a frozen pharmaceutical composition prepared from cultured human mesenchymal stem cells and a pharmaceutical composition containing human mesenchymal stem cells. This method is a method for producing a frozen pharmaceutical composition containing human mesenchymal stem cells, which comprises the following steps in this order: (a) adding trypsin to human mesenchymal stem cells in a culture vessel to detach the cells from the surface of the culture vessel; (b) adding a bicarbonate Ringer's solution containing human serum albumin to the detached cells to terminate the reaction with trypsin, and washing the cells with the bicarbonate Ringer's solution containing human serum albumin; (c) suspending the cells in a bicarbonate Ringer's solution containing human serum albumin and dimethyl sulfoxide; (d) putting the resulting suspension in a container which allows freezing of what is contained therein, and sealing the container; and (e) freezing the suspension put in the container.

## Claims

### Claim 1

A pharmaceutical composition which comprises human mesenchymal stem cells in a bicarbonate Ringer's solution containing human serum albumin, dimethyl sulfoxide, 120-150 mEq/L of sodium ion, 3.6-4.4 mEq/L of potassium ion, 2.7-3.3 mEq/L of calcium ion, 0.9-1.1 mEq/L of magnesium ion, 100-125 mEq/L of chloride ion, 22-28 mEq/L of bicarbonate ion, and 4.5-5.5 mEq/L of citrate ion.

### Claim 2 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the osmotic pressure ratio of the solution to the physiological saline is 0.9-1.1, and the pH of the solution is 6.8-7.8.

### Claim 3 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the concentration of human serum albumin is 3-8 W/V %.

### Claim 4 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the concentration of dimethyl sulfoxide is 8-12 W/V %.

### Claim 5 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the human mesenchymal stem cells are those derived from human bone marrow.

### Claim 6 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the composition contains the human mesenchymal stem cells at a density of 1×105-1×108 cells/mL.

### Claim 7 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the composition contains the human mesenchymal stem cells at a density of 1×106-1×107 cells/mL.

### Claim 8 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the composition is held in a sealed container which can allow freezing of what is contained therein.

### Claim 9 (depends on 8)

The pharmaceutical composition according to claim 8, wherein the composition is held in the sealed container at a volume of 1-30 mL.

### Claim 10 (depends on 1)

The pharmaceutical composition according to claim 1, wherein the composition is in a frozen state.

### Claim 11

A method for preparation of human mesenchymal stem cells free of the medium used for their culture comprising collecting the human mesenchymal stem cells from a culture vessel in which they were allowed to proliferate, which method further comprises the steps:
(a) adding trypsin to human mesenchymal stem cells in the culture vessel to detach the cells from the surface of the culture vessel, and
(b) adding a bicarbonate Ringer's solution containing human serum albumin to the detached human mesenchymal stem cells to terminate the reaction with trypsin while washing the cells with the bicarbonate Ringer's solution containing human serum albumin, 120-150 mEq/L of sodium ion, 3.6-4.4 mEq/L of potassium ion, 2.7-3.3 mEq/L of calcium ion, 0.9-1.1 mEq/L of magnesium ion, 100-125 mEq/L of chloride ion, 22-28 mEq/L of bicarbonate ion, and 4.5-5.5 mEq/L of citrate ion.

### Claim 12 (depends on 11)

The method for preparation according to claim 11, wherein the, the osmotic pressure ratio of the solution to the physiological saline is 0.9-1.1, and the pH of the solution is 6.8-7.8.

### Claim 13 (depends on 11)

The method for preparation according to claim 11, wherein the concentration of human serum albumin in the bicarbonate Ringer's solution in step (b) is 0.5-3 W/V %.

### Claim 14 (depends on 11)

A method for production of a frozen pharmaceutical composition comprising human mesenchymal stem cells, which method comprises the steps in this order:
(c) suspending the human mesenchymal stem cells prepared by a method of claim 11 above in a bicarbonate Ringer's solution containing human serum albumin, dimethyl sulfoxide, 120-150 mEq/L of sodium ion, 3.6-4.4 mEq/L of potassium ion, 2.7-3.3 mEq/L of calcium ion, 0.9-1.1 mEq/L of magnesium ion, 100-125 mEq/L of chloride ion, 22-28 mEq/L of bicarbonate ion, and 4.5-5.5 mEq/L of citrate ion,
(d) putting the resulting suspension obtained by step (c) in a container which allows freezing of what is contained therein, and sealing the container, and
(e) freezing the suspension put in the container in above (d).

### Claim 15 (depends on 14)

The method for production according to claim 14, wherein the concentration of human serum albumin in the bicarbonate Ringer's solution in step (c) is 3-8 W/V %.

### Claim 16 (depends on 14)

The method for production according to claim 14, wherein the concentration of dimethyl sulfoxide in the bicarbonate Ringer's solution in step (c) is 8-12 W/V %.

### Claim 17 (depends on 14)

The method for production according to claim 14, wherein the human mesenchymal stem cells are suspended at a density of 1×105-1×108 cells/mL in step (c).

### Claim 18 (depends on 14)

The method for production according to claim 14, wherein the human mesenchymal stem cells are suspended at a density of 1×106-1×107 cells/mL in step (c).