成人骨髓间充质干细胞体外培养方法的建立及生长特性分析

目的 建立成人骨髓间充质干细胞(marrow mesenchymal stem cells,MMSCs)体外分离培养和扩增方法,观察MMSCs在体外培养的生物学特性,为MMSCs的临床应用奠定技术基础.方法 采集成人骨髓,密度梯度法分离单个核细胞,利用DMEM/F12培养基添加自体血清进行黏附培养和扩增MMSCs,光镜观察MMSCs生长和形态变化,计数和MTT法观察原代和传代培养的增殖及生长特征,免疫组化法分析MMSCs纯度.结果 MMSCs贴壁生长,呈典型MMSCs形态和生长特征,原代及传代5代内的MMSCs均有活跃的增殖能力,其中CD105阳性细胞＞98%.结论 密度梯度离心结合贴壁培养法可获得高纯度和高增殖活性的MMSCs,成人MMSCs至少在传代培养5代内生长旺盛并维持其生物特性,可满足临床治疗和人体生物组织研究要求.

Growth and biological character of mesenchymal stem cells derived from adult human marrow in vitro

[Objective] To establish the in vitro culture and expense methods of adult marrow mesenchymal stem cells(MMSCs) for clinic therapy and regeneration medical study and analyze the biological character of cultured MMSCs. [Methods] Bone marrow (10 mL) was obtained from the iliac crest of healthy voluntary donors in 20~30 years (10 mL) was obtained from the iliac crest of healthy voluntary donors in 20~30 years old after informed consent. MMSCs were isolated and purified by centrifuge and adherent culuture in vitro. The proliferation and growth characteristics were investigated in primary and passage culture by morphology and cell number observe and MTT essay. MSCs Phenotypical markers were analyzed by immunochemistry. [Results] Adult human MMSCs growth into uniform fibroblast-like cells rapidly in plastic cell cultural board and mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity from original passage to passage 5. The passage MMSCs growth were faster than the original cells. Immunochemistry results showd that the cultured cell positive for CD105 and negative for CD34. [Conclusion] Adult human MMSCs can be cultured and extensively proliferate and retain biological feature for at least for 5 passage. The methods of MMSCs culture can be used for for clinic MSCs therapy and related research.