# US 12,215,346 B2 · Method for isolating and culturing neural stem cells with high efficiency

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- Number: US 12,215,346 B2
- Document ID: US12215346B2
- Status: Granted
- Kind: B2
- Application number: 16/980,889
- Filed: 2019-01-30
- Published: 2022-08-04
- Granted: 2025-02-04
- Earliest claimed: 2018-03-16
- Assignees: Medinno Inc.
- Applicants: Medinno Inc.
- Inventors: Kyeung Min Joo; Hyun Nam
- Web page: https://patentgrep.com/patent/US12215346B2

## CPC

- C12N 5/0619: {Neurons}
- C12N 5/0623: {Stem cells}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/63: {derived from plants}
- C12Y 301/21001: Deoxyribonuclease I (3.1.21.1)
- C12Y 304/22002: Papain (3.4.22.2)
- G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition}
- 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)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to a method for isolating and culturing neural stem cells with high efficiency, which may shorten the time for isolation and culture by simplifying a method for isolating and culturing neural stem cells and may increase the acquisition yield of neural stem cells. The present invention provides a method for isolating and culturing neural stem cells with high efficiency, comprising the steps of adding brain tissue into an enzyme solution so as to subject the brain tissue to enzyme treatment; physically isolating cell clumps from the enzyme treated brain tissue by dividing the cell clumps according to size and removing impurities; and inoculating the cell clumps on a culture dish so as to subculture.

## Claims

### Claim 1

A method for isolating and culturing neural stem cells with high efficiency, comprising:
placing brain tissue in an enzyme solution for enzyme treatment;
physically dissociating cell clumps from the enzyme-treated brain tissue;
separating the cell clumps by size and removing impurities; and
performing subculture by seeding the cell clumps that have a size greater than 70 μm and less than 100 μm in a culture dish,
wherein the method of isolating and culturing neural stem cells does not comprise isolating single cells,
wherein the step of separating the cell clumps by size comprises the steps of:
a) filtering the cell clumps through a 100 μm mesh and collecting the filtrate;
b) filtering the filtrate of step a) through a 70 μm mesh; and
c) collecting the cell clumps caught by the 70 μm mesh.

### Claim 2 (depends on 1)

The method of claim 1, wherein the enzyme solution contains papain; DNase I; and D, L-cysteine.

### Claim 3 (depends on 1)

The method of claim 1, wherein the brain tissue is adult brain tissue.