无动物源成分培养基用于人脐带间充质干细胞培养的研究

Culture of human umbilical cord mesenchymal stem cells by Xeno/serum-free medium

目的建立一种无动物源成份无血清的间充质干细胞培养基XF/SF-MSCM,并探讨其支持人脐带间充质干细胞(h UC-MSCs)体外增殖、维持分化潜能的效果。方法组织块贴壁法分离获得原代人脐带间充质干细胞,P1代起分别使用自制的XF/SF-MSCM培养基与含10%胎牛血清的传统培养基(SC-MSCM)对脐带间充质干细胞进行连续培养传代至P6代,观察比较两组P6代细胞的形态及增殖能力,以流式细胞术检测连续传代后细胞的免疫学表型,比较其成骨、成脂肪的分化潜能。结果分离获得的原代P0脐带间充质干细胞,分别在XF/SFMSCM与SC-MSCM两种培养基中连续培养传代,细胞增殖能力及形态无显著差别。两组P6代细胞的生长曲线相似,但XF/SF-MSCM组间充质干细胞贴壁的紧密程度稍低;流式细胞检测结果显示,XF/SF-MSCM组细胞保持了间充质干细胞的免疫学表型,高表达CD29、CD44、CD90,不表达CD31、CD34、CD45并维持了良好的成骨和脂肪细胞的分化能力。结论本室制备的无动物源成份无血清培养基XF/SF-MSCM可支持脐带间充质干细胞的体外扩增,维持其免疫学表型及分化潜能,提供数量充足的高质量间充质干细胞,满足临床治疗及生物医学研究的需要。

Objective To establish a xeno/serum-free medium for human umbilical cord mesenchymal stem cells（h UC-MSCs） and evaluate its capacity to support the expansion of h UC-MSCs in vitro.Methods Primary h UC-MSCs were isolated from Wharton＇s Jelly of umbilical cord of healthy newborn.Cells were respectively maintained and consecutively passaged in our homemade xeno/serum-free medium（XF/SF-MSCM） and conventional serum containing medium（SC-MSCM） as a control from passage1（P1） to passage 6（P6）.Cell morphology and proliferation were observed and compared.The immunological phenotype of P6 h UC-MSCs was analyzed by Flow Cytometry and the differentiation potency was also investigated.Results There is no significant difference in proliferation rate and morphology between h UC-MSCs cultured in X/SF-MSCM and SC-MSCM.The growth curve of P6 cells was similar when they were cultured in two culture systems.The P6 cells cultured in XF/SF-MSCM showed MSCs specific surface marker expression phenotype：CD29＋,CD44＋,CD90＋ and CD31-,CD34-,CD45-.Furthermore,P6 cells maintained in XF/SF-MSCM could be induced to adipocyte and osteoplastic differentiation.Conclusion Our homemade XF/SF-MSCM for human umbilical cord mesenchymal stem cells（h UC-MSCs） can efficiently support the sequential passage and expansion of h UC-MSCs in vitro and maintain the differentiation potentials.The XF/SF-MSCM medium provides an efficient amplification system to produce sufficient and high quality MSCs for either clinical studies or applications.