	US 12,215,346 B2
	Method for isolating and culturing neural stem cells with high efficiency
Kyeung Min Joo, Seoul (KR); and Hyun Nam, Gyeonggi-do (KR)
Assigned to MEDINNO INC., Gyeonggi-do (KR)
Appl. No. 16/980,889
Filed by MEDINNO INC., Gyeonggi-do (KR)
PCT Filed Jan. 30, 2019, PCT No. PCT/KR2019/001288 § 371(c)(1), (2) Date Sep. 15, 2020, PCT Pub. No. WO2019/177270, PCT Pub. Date Sep. 19, 2019.
Claims priority of application No. 10-2018-0031101 (KR), filed on Mar. 16, 2018; and application No. 10-2019-0011226 (KR), filed on Jan. 29, 2019.
Prior Publication US 2022/0243175 A1, Aug. 4, 2022
Int. Cl. C12N 5/0797 (2010.01); C12N 9/22 (2006.01); C12N 9/50 (2006.01); G01N 1/40 (2006.01)

CPC C12N 5/0623 (2013.01) [C12N 9/22 (2013.01); C12N 9/63 (2013.01); C12Y 301/21001 (2013.01); C12Y 304/22002 (2013.01); G01N 1/4044 (2013.01); C12N 2509/00 (2013.01)]

3 Claims

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.