# US 10,870,833 B2 · Method for separating cell from biological tissue

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

- Number: US 10,870,833 B2
- Document ID: US10870833B2
- Status: Granted
- Kind: B2
- Application number: 16/109,242
- Filed: 2018-08-22
- Published: 2018-12-20
- Granted: 2020-12-22
- Earliest claimed: 2013-02-01
- Assignees: Tohoku University; Meui Seika Pharma Co., Ltd.; National University Corporation Tokyo University of Agriculture and Technology
- Applicants: Tohoku University; Meiji Seika Pharma Co., Ltd.; National University Corporation Tokyo University of Agriculture and Technology
- Inventors: Masafumi Goto; Kazutaka Murayama; Youhei Yamagata; Kimiko Watanabe
- Web page: https://patentgrep.com/patent/US10870833B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 5/067: {Hepatocytes}
- C12N 2509/00: Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided is a method for efficiently and stably separating cells having a high biological activity from a biological tissue, by using a degrading-enzyme composition, which is prepared by adding an enzyme for degrading a major protein of the biological tissue in an amount determined depending on the composition of the major protein to a predetermined amount of a neutral protease and/or a protease derived from Clostridium sp. According to this method, the type and amount of protein-degrading enzyme to be used for isolating cells can be determined from the composition of a major protein of the biological tissue. Thus, cells having a high biological activity can be efficiently separated while reducing the amount of protein-degrading enzyme to be used.

## Claims

### Claim 1

A method for separating pancreatic islets from a pancreas tissue, using a degrading-enzyme composition, comprising:
measuring an abundance of collagen I and collagen III in the pancreas tissue,
determining an amount ratio of collagenase H to collagenase G derived from Clostridium sp. based on the measured abundance of collagen I and collagen III, wherein a higher amount ratio is used when the measured abundance is higher and a lower amount ratio is used when the measured abundance is lower,
preparing the degrading-enzyme composition by adding collagenase H and collagenase G derived from Clostridium sp. with the determined ratio, to a predetermined amount of one or more of a neutral protease or a protease derived from Clostridium sp.,
treating the pancreas tissue with the degrading-enzyme composition, and
separating pancreatic islets from the pancreas tissue treated with the degrading-enzyme composition,
wherein collagenase H degrades collagen I and collagen III, and
wherein the amount ratio (H/G) of collagenase H to collagenase G in the degrading-enzyme composition is 0.35 or more.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the abundance of collagen I and collagen III in the pancreas tissue is measured by enzymatic immunoassay using specific antibodies against collagen I and collagen III.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the neutral protease is thermolysin or a neutral protease derived from Clostridium sp.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the degrading-enzyme composition contains thermolysin 0.3 mg/10 ml, collagenase G 2 mg/10 ml and collagenase H 1.1 mg/10 ml.

### Claim 5 (depends on 3)

The method according to claim 3, wherein the degrading-enzyme composition contains thermolysin 0.3 mg/10 ml, collagenase G 8.4 mg/10 ml and collagenase H 2.9 mg/10 ml.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the degrading-enzyme composition contains a neutral protease derived from Clostridium sp., as the neutral protease, and a protease having an α-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradation activity, as the protease derived from the Clostridium sp.