US 12,480,092 B2
Method for preparing mature red blood cells in vitro using peripheral blood
Xiaofei Gao, Hangzhou (CN); Yanjie Huang, Hangzhou (CN); and Qing Zhang, Hangzhou (CN)
Assigned to WESTLAKE THERAPEUTICS (HANGZHOU) CO. LIMITED, Hangzhou (CN)
Appl. No. 17/430,020
Filed by WESTLAKE THERAPEUTICS (HANGZHOU) CO. LIMITED, Hangzhou (CN)
PCT Filed Feb. 26, 2020, PCT No. PCT/CN2020/076820
§ 371(c)(1), (2) Date Aug. 11, 2021,
PCT Pub. No. WO2020/173466, PCT Pub. Date Sep. 3, 2020.
Claims priority of application No. 201910151896.6 (CN), filed on Feb. 28, 2019.
Prior Publication US 2022/0112463 A1, Apr. 14, 2022
Int. Cl. C12N 5/078 (2010.01)
CPC C12N 5/0641 (2013.01) [C12N 2500/24 (2013.01); C12N 2500/32 (2013.01); C12N 2501/125 (2013.01); C12N 2501/14 (2013.01); C12N 2501/2303 (2013.01); C12N 2501/2306 (2013.01); C12N 2501/33 (2013.01); C12N 2501/727 (2013.01); C12N 2506/115 (2013.01)] 10 Claims
OG exemplary drawing
 
1. A method for producing red blood cells (RBCs), comprising:
(a) collecting lineage and CD34 negative cells (Lin−CD34− cells) from a peripheral blood sample,
(b) expanding the Lin−CD34− cells; and
(c) inducing the expanded Lin−CD34− cells to differentiate into mature red blood cells, wherein step (b) comprises culturing the Lin−CD34− cells in a hematopoietic stem cell expansion medium supplemented with a combination of cytokines, and wherein the combination of cytokines comprises fms-like tyrosine kinase 3 ligand (Flt3L), stem cell factor (SCF), interleukin 3 (IL-3), and interleukin 6 (IL-6),
wherein step (c) comprises:
(c1) in a first differentiation medium supplemented with cytokines related to erythroid development, culturing the expanded Lin+CD34− cells to induce the same to differentiate into erythroid cells, wherein the cytokines related to erythroid development include IL-3 and SCF, wherein the first differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing about 5-15% (v/v) FBS, about 2-10% (v/v) human plasma, about 1-4 mM glutamine, about 5-15 mg/ml BSA, about 300-600 μg/mL human transferrin, about 8-13 μg/mL human insulin, 1-3% Penicillin-Streptomycin, about 3-7 ng/ml human IL-3, about 4-7 U/mL human EPO, and about 80-120 ng/mL human SCF; and
(c2) culturing the erythroid cells in a second differentiation medium to induce enucleation, wherein the second differentiation medium lacks the cytokines related to erythroid development as compared to the first differentiation medium, wherein the second differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing about 5-15% FBS, about 2-10% human plasma, about 1-4 mM glutamine, about 5-15 mg/ml BSA, about 300-600 μg/mL human transferrin, about 8-13 μg/mL human insulin, an effective amount of one or more antibiotics, and about 1-5 U/mL human EPO.