Probe for analyzing biological tissue and method for utilizing same

Patent metadata

Field	Value
Publication number	US-9354240-B2
Application number	US-201214003495-A
Country	US
Kind code	B2
Filing date	Mar 15, 2012
Priority date	Mar 16, 2011
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

The present invention relates to a method for obtaining cells or cell populations having a high biological activity from a biological tissue by enzymatic isolation, and probes for use in the method; more specifically to a method for analyzing a biological tissue, comprising applying two or more probes respectively containing biological-component binding domains through which two or more proteins bind to a predetermined biological component, to an isolated biological tissue, and analyzing binding amounts of the probes to the biological tissue.

First claim

The invention claimed is:

1. A method for separating cells or cell populations from a biological tissue, comprising:
applying two or more probes respectively containing biological-component binding domains of two or more proteases and two or more visualization molecules to an isolated biological tissue,
measuring binding amounts of the probes to the biological tissue through the visualization molecules,
determining a quantitative ratio of the proteases based on results of the measurement,
applying the proteases in the quantitative ratio to the biological tissue, and
separating the cells or the cell populations from the biological tissue,
wherein each of the proteases is selected from the group consisting of collagenase, trypsin, chymotrypsin, dispase, elastase, papain, pronase, thermolysin, subtilisin, bromelain, phytin, and thermitase, and
wherein each of the two or more visualization molecules is a molecule that is different from the other two or more visualization molecules and that is selected from the group consisting of fluorescent molecules, luminescent molecules, and radioisotopes including a positron nuclide.

2. The method according to claim 1 , wherein at least one of the two or more visualization molecules is a luciferase protein and/or fluorescent molecule selected from the group consisting of GFP, EGFP, YFP, BFP, CFP, DsRED, tdTomato, and RFP.

3. The method according to claim 2 , wherein the luciferase protein has a peak wavelength and luminescent intensity different from a wild-type luciferase.

4. The method according to claim 2 , wherein the luciferase protein is linked to a fluorescent molecule selected from the group consisting of GFP, EGFP, YFP, BFP, CFP, DsRED, tdTomato, and RFP via a linker.

5. The method according to claim 1 , wherein each of the biological-component binding domains and the visualization molecules form a fusion protein.

6. The method according to claim 1 , wherein each of the biological-component binding domains comprises a collagen binding domain of the proteases.

7. The method according to claim 6 , wherein the collagen binding domains are collagen binding domains of collagenases selected from collagenases derived from the genus Clostridium.

8. The method according to claim 7 , wherein the collagen binding domains are collagen binding domains of collagenase G and collagenase H derived from Clostridium histolyticum.

9. The method according to claim 1 , comprising: applying the two or more probes separately to a biological tissue, and measuring binding amounts of the probes separately.

10. The method according to claim 1 , comprising: applying the two or more probes simultaneously to a biological tissue, and measuring binding amounts of the probes simultaneously.

11. The method according to claim 1 , wherein the probes each contain 1 to 20 repeats of a biological-component binding domain.

12. A method for separating cells or cell populations from a biological tissue, comprising:
applying two or more probes respectively containing collagen binding domains of two or more collagenases and two or more visualization molecules to an isolated biological tissue,
measuring binding amounts of the probes to the biological tissue through the visualization molecules,
determining a quantitative ratio of the collagenases based on results of the measurement,
applying the collagenases in the quantitative ratio to the biological tissue, and
separating the cells or the cell populations from the biological tissue,
wherein each of the two or more visualization molecules is a molecule that is different from the other two or more visualization molecules and that is selected from the group consisting of fluorescent molecules, luminescent molecules, and radioisotopes including a positron nuclide.

13. The method according to claim 12 , wherein at least one of the two or more visualization molecules is a luciferase protein and/or fluorescent molecule selected from the group consisting of GFP, EGFP, YFP, BFP, CFP, DsRED, tdTomato, and RFP.

14. The method according to claim 12 , wherein the collagen binding domains are collagen binding domains of collagenases selected from collagenases derived from the genus Clostridium.

15. The method according to claim 14 , wherein the collagen binding domains are collagen binding domains of collagenase G and collagenase H derived from Clostridium histolyticum.

Assignees

• Yamagata Youhei
• Goto Masafumi
• Watanabe Kimiko
• Univ Tohoku
• Meiji Seika Pharma Co Ltd

Inventors

• Yamagata Youhei
• Goto Masafumi
• Watanabe Kimiko

Classifications

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• G01N33/56966 Primary

  Animal cells · CPC title

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C07K14/43595

  from coelenteratae, e.g. medusae · CPC title

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title