Method for separating cell from biological tissue

Patent metadata

Field	Value
Publication number	US-10870833-B2
Application number	US-201816109242-A
Country	US
Kind code	B2
Filing date	Aug 22, 2018
Priority date	Feb 1, 2013
Publication date	Dec 22, 2020
Grant date	Dec 22, 2020

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.

First claim

The invention claimed is:

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.

2. 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.

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

4. 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.

5. 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.

6. 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.

Assignees

• Univ Tohoku
• Meiji Seika Pharma Co Ltd
• National Univ Corporation Tokyo Univ Of Agriculture And Technology
• Meui Seika Pharma Co Ltd

Inventors

• Goto Masafumi
• Murayama Kazutaka
• Yamagata Youhei
• Watanabe Kimiko

Classifications

• C12N5/067 Primary

  Hepatocytes · CPC title

• G01N33/56966

  Animal cells · CPC title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title

• C12N5/0676 Primary

  Pancreatic cells · CPC title

• C12N5/00

  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) · CPC title