	US 11,946,073 B2
	Method for non-enzymatic 3D culture and amplification of mesenchymal stem cells
Bin Zheng, Tianjin (CN); Yulin Cao, Beijing (CN); Wenchang Peng, Tianjin (CN); and Shixiang Cheng, Tianjin (CN)
Assigned to Healthina Stem Cell Industry Platform (Tianjin) Limited, Tianjin (CN); and Tangyi Holdings(Shenzhen) Limited, Shenzhen (CN)
Filed by Healthina Stem Cell Industry Platform (Tianjin) Limited, Tianjin (CN); and Tangyi Holdings(Shenzhen) Limited, Shenzhen (CN)
Filed on May 8, 2022, as Appl. No. 17/739,127.
Claims priority of application No. 202210226396.6 (CN), filed on Mar. 9, 2022.
Prior Publication US 2023/0287352 A1, Sep. 14, 2023
Int. Cl. C12N 5/0775 (2010.01); A61K 9/16 (2006.01); C12N 5/00 (2006.01)

CPC C12N 5/0663 (2013.01) [A61K 9/1647 (2013.01); A61K 9/1652 (2013.01); A61K 9/1694 (2013.01); C12N 5/0075 (2013.01)]

4 Claims

1. A method for non-enzymatic 3D culture and amplification of mesenchymal stem cells (MSCs), comprising the following steps:

S1) preparing poly(lactic-co-glycolic acid) (PLGA) porous microspheres, wherein preparing the PLGA porous microspheres comprises the following steps:

S1a) dissolving PLGA, cholesterol, and NH₃HCO₃in ether at a weight ratio of (10:1:1)-(10:2:2) at a room temperature to obtain an ether mixture,

adding water to the ether mixture, wherein a volume ratio of the water added to the ether is (3:1)-(4:1), and

performing ultrasonication for 15-40 min to form a water-in-oil (W/O) emulsion; and

S1b) performing spray drying on the W/O emulsion obtained in step (Sla) by a spray dryer to form the PLGA porous microspheres;

S2) preparing a PLGA-PEG-PLGA thermosensitive coating microcarrier, wherein PEG is polyethylene glycol;

S3) culturing and amplifying the MSCs; and

S4) performing non-enzymatic separation of the MSCs: reducing a culture temperature to below a critical phase transition temperature, and centrifuging a culture medium to collect the MSCs.