脐带血间充质干细胞体外分离、纯化及培养

目的　研究脐带血 (HUCB)中含有的间充质干细胞 (MSCs)体外分离、纯化及培养条件 ,探究其作为种子细胞应用于实验和临床的可行性。方法　无菌条件下取正常足月剖腹产的脐带血 ,经肝素抗凝 ,用淋巴细胞分离液分离脐血的单个核细胞 ,以偏酸性的MesencultTM作为培养基进行培养和纯化获得贴壁细胞层 ,用流式细胞仪检测MSCs表面抗原。结果　来源于脐血的单个核细胞经体外培养贴壁后出现破骨样及间充质样的细胞 ,间充质细胞为成纤维样的细胞形态 ,并表达MSCs相关的抗原 (CD2 9、CD4 4、CD1 6 6 ) ,但不表达造血细胞抗原 (CD34 、CD4 5) ,这与源于骨髓的MSCs一致。结论　脐带血来源的MSCs能在体外培养、扩增 ,而用于满足实验和临床的需要

THE ISOLATION PURIFICATION AND CULTURE OF HUCA MSCs

Objective: To investigate the isolation, purification and culture of human umbilical cord blood(HUCB) mesenchymal stem cells(MSCs) in vitro, and to observe the feasibility of using MSCs as seed cells in experiment and clinic. Methods: HUCB were collected from full term deliveries scheduled for cesarean section, all specimens was obtained sterilely with preservative-free heparin, the cord blood mononuclear cell was isolated by lymphocyte separation medium, and purified and culture with Mesencult TM medium and acidic environment to produce adherent layer, the surface antigen expression of MSCs was detected by flow cytometry. Results: The UCB-derived mononuclear cells, when set in culture, gave rise to adherent cells, which exhibited either an osteoclast-or mesenchymal-like phenotype, cells with the MSCs displayed a fibroblast-like morphology and expressed several MSCs-related antigens (CD 29, CD 44, CD 166), but did not express haematopoietic cells antigens(CD 34, CD 45), which was identical to human bone marrow-derived MSCs. Conclusion: MSCs in HUCB can culture and expand in vitro, which could be regarded as an alternative source of MSCs for experimental and clinical needs.