In vitro culture and influencing factors of mesenchymal stem cells derived from human umbilical blood
- VernacularTitle:人脐带血间充质干细胞的体外培养及其影响因素
- Author:
Wei HUANG
;
Jianzhong ZHU
;
Zongning MIAO
;
Ling WANG
;
Xin WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(3):572-575
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Multipotential mesenchymal stem cells (MSCs) can be differentiated into bone, fat, cartilage, muscle and endothelial cell at the specific conditions, so it draws great attentions of the related study.OBJ ECTTVE: To establish the method of in vitro culture and expansion of human umbilical blood-derived MSCs and investigate their influencing factors.DESTGN: Randomized and controlled trials.SETTING: Department of Tissue Engineering and Cell Biology in Wuxi Third People's Hospital.MATERTALS: Seventy cases of human umbilical cord blood (HUCB) of healthy neonates (selected from Parturient Room, Department of Gynecology and Obstetrics, Wuxi Third People's Hospital, with the informed consents of the puerpera and their relatives), 40 mL each; dulbecco's minimal essential medium (DMEM) of low glucose (LG) at the dose of 50 g/L, fetal bovine serum (FBS), mycillin and trypsin (all were from Gibco), FITC-labeled mice anti-human CD29, CD105 and CD106 (Ancell) monoclonal fluorescent antibody, PE-labeled mice anti-human CD34, CD44, CD45, CD19, HLA-DR (Immunotech)monoclonal fluorescent antibody, Ficoll separation medium (Pharmacia).METHODS: The experiment was carried out in Department of Tissue Engineering and Cell Biology in Wuxi Third neonatal cord blood were anticoagulated by 25 u/mL heparin, and 60 cases of isolated HUCB mononuclear cell were precipitated and suspended by cell culture fluid (50 g/L DMEM-LG + 50 g/L FBS + 10 g/L mycillin), while other 10 cases were purified with DMEM of high glucose (100 g/L) to produce adherent layer. Once the cells reached fluences of FBS coating on adhesive rats of MSCs: Some of the purified HUCB MSCs at logarithmic phase were growth curve of MSCs: The growth of cells were observed by phase contrast microscope (OLYMPUS CK40), and were detected by EPICS-ALTRA flow cytometry.FBS coating and without FBS coating at the different time points, and their adhesive rates.RESULTS: All 70 cases of HUCB MSCs were adopted in this study, however, 10 samples failed to obtain the ideal MSCs cord blood MSCs after culture. During 2 weeks of culture, primary cells began to double for 3-4 days and reached conand CD105, but did not express antigens CD34, CD45, CD106 and HLA-DR, which was identical to surface labeling of those without FBS coating (P＜0.01).CONCLUSrON: MSCs in HUCB can be cultured and expanded in vitro, and are regarded as an alternative source of MSCs for experimental and clinical applications. Moreover, high glucose can depress the growth and proliferation of MSCs in HUCB.
