US 11,898,164 B2
Method for producing pluripotent stem cell-derived spherical neural mass having suppressed teratoma formation
Myung Soo Cho, Seoul (KR); Mi Sun Lim, Gyeonggi-do (KR); and So Yeon Ji, Chungcheongbuk-do (KR)
Assigned to S.BIOMEDICS CO., LTD., Seoul (KR)
Appl. No. 16/474,662
Filed by JEIL PHARMACEUTICAL CO., LTD., Seoul (KR)
PCT Filed Dec. 20, 2017, PCT No. PCT/KR2017/015110
§ 371(c)(1), (2) Date Jun. 28, 2019,
PCT Pub. No. WO2018/124605, PCT Pub. Date Jul. 5, 2018.
Claims priority of application No. 10-2016-0184325 (KR), filed on Dec. 30, 2016.
Prior Publication US 2019/0330590 A1, Oct. 31, 2019
Int. Cl. C12N 5/00 (2006.01); C12N 5/0793 (2010.01); C12N 5/0797 (2010.01)
CPC C12N 5/0619 (2013.01) [C12N 5/0623 (2013.01); C12N 2501/115 (2013.01); C12N 2506/08 (2013.01); C12N 2506/45 (2013.01); C12N 2533/20 (2013.01); C12N 2533/52 (2013.01); C12N 2533/54 (2013.01); C12N 2533/90 (2013.01)] 10 Claims
 
1. A method for producing spherical neural masses having suppressed teratoma formation, the method including the following steps:
1) fragmentation of the spherical neural masses to obtain fragmented spherical neural masses;
2) adhesion-culturing of the fragmented spherical neural masses in a culturing dish comprising culturing medium for neuronal progenitor cell to obtain adhesion-cultured spherical neural masses;
3) performing single-cellularization of the adhesion-cultured spherical neural masses by adding enzyme to obtain single cells;
4) inducing spontaneous aggregation of cells by adhesion-culturing of the single cells of step 3) in a culturing dish without an additional substrate coated thereon and without adding a differentiation-inducing factor to obtain aggregates attached to a bottom of the culturing dish;
5) removing floating cells after step 4) and inducing spontaneous re-aggregation of single cells by repeatedly suspending the aggregates attached to the bottom of culturing dish to obtain re-aggregated spherical neural masses; and
6) recovering the spherical neural masses re-aggregated in step 5), wherein the re-aggregated spherical neural masses contain fewer undifferentiated cells compared to the spherical neural masses in step 1).