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Isolation and culture of MB-MSCs

YL Yonghai Li
HG Haiyao Gao
TB Tobias M. Brunner
XH Xiaoxi Hu
YY Yushan Yan
YL Yanli Liu
LQ Liang Qiao
PW Peihua Wu
ML Meng Li
QL Qing Liu
FY Fen Yang
JL Juntang Lin
ML Max Löhning
PS Ping Shen
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Menstrual blood samples were donated by healthy volunteers during the first few days of menses. Upon collection, blood samples were subjected to standard Ficoll gradient centrifugation. The mononuclear cells at the interlayer were harvested together with the suspending deciduous endometrium and plated in T25 flasks with complete DMEM medium supplemented with 15% FBS and 100 U/ml penicillin/streptomycin. After two days of culture, culture medium was replaced to remove non-adherent cells. MB-MSCs remained as adherent cells grown into visible colonies. Medium was renewed every 3 days, and MB-MSCs were subcultured to new flasks when they reached 80% confluence (P0). Cells were then either directly used for experiments or cultured further till passage 3 and frozen in liquid nitrogen. The entire procedures were performed with consent of the donors and approved by the Ethics Committee of Xinxiang Medical University, China.

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