# US 9,040,300 B2 · Method of washing adherent cell using trehalose-containing cell-washing solution

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- Number: US 9,040,300 B2
- Document ID: US9040300B2
- Status: Granted
- Kind: B2
- Application number: 14/035,100
- Filed: 2013-09-24
- Published: 2014-04-03
- Granted: 2015-05-26
- Earliest claimed: 2012-09-28
- Assignees: Otsuka Pharmaceutical Factory, Inc.
- Applicants: Otsuka Pharmaceutical Factory, Inc.
- Inventors: Eiji Kobayashi; Tamaki Wada; Masako Doi
- Web page: https://patentgrep.com/patent/US9040300B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- C12N 2500/34: Sugars
- C12N 2501/734: Proteases (EC 3.4.)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods of washing adherent cells, capable of effectively suppressing cell death due to proteolytic enzyme treatment for detaching the adherent cell from a culture vessel and subsequent cell treatment; cell-washing solutions used for the washing method; methods of producing cell suspensions for transplantation using the cell-washing solution; and kits comprising the cell-washing solution. Trehalose or its derivative or a salt thereof is added to physiological aqueous solutions to prepare cell-washing solutions containing trehalose or its derivative or a salt thereof as an active ingredient. The cell-washing solutions can be used to wash adherent cells before detaching the adherent cells from a culture vessel by proteolytic enzyme treatment to suppress cell death due to the proteolytic enzyme treatment. The concentration of trehalose applied to the cell-washing solution may be a concentration capable of suppressing the cell death due to the proteolytic enzyme treatment, such as 0.1 to 20 (w/v) %.

## Claims

### Claim 1

A method of producing a cell suspension for transplantation, comprising:
(a) removing medium from a culture vessel to which an adherent cell is adhered and washing the adherent cell with a cell-washing solution comprising a physiological aqueous solution with trehalose or its derivative or a salt thereof added thereto to obtain a washed adherent cell;
(b) detaching the washed adherent cell from the culture vessel by proteolytic enzyme treatment;
(c) suspending the adherent cell detached from the culture vessel in a protein-containing physiological aqueous solution to make a cell suspension; and
(d) removing the solution in the cell suspension and washing the adherent cell with the physiological aqueous solution.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the adherent cell is a mesenchymal stem cell.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the cell-washing solution of (a) comprises trehalose or its derivative or a salt thereof in a concentration of 1 to 15 (w/v) %.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the proteolytic enzyme of (b) is trypsin.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the protein-containing physiological aqueous solution of
(c) further comprises trehalose or its derivative or a salt thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the protein-containing physiological aqueous solution of (c) is a serum-containing basal medium for animal cell culture.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the physiological aqueous solution of (d) further comprises trehalose or its derivative or a salt thereof.