人血小板裂解液替代胎牛血清大规模扩增人脐带间充质干细胞

背景：目前国内外大多数实验仍采用经典的培养基和胎牛血清进行脐带间充质干细胞培养，血清培养潜在的不安全因素限制了未来临床应用的可行性。目的：以人血小板裂解液替代胎牛血清培养、鉴定人间充质干细胞，以期进一步应用于临床低密度扩增人间充质干细胞。方法：人血小板裂解液通过反复冻融、离心、过滤、浓缩等方法制备。以 IMDM 为基础培养基，添加5%浓缩血小板裂解液作为实验组培养基；以添加体积分数10%胎牛血清为对照组培养基。采用酶消化法分离培养人脐带间充质干细胞，以3000/cm2的浓度进行传代培养，待细胞扩增至P5代后，进行细胞形态与直径、免疫表型、成骨成脂分化、克隆形成率等细胞生物学特性检测，并比较两者之间的差别。结果与结论：人血小板裂解液扩增的脐带间充质干细胞形态细长，有更高的细胞累积群倍数；克隆形成检测结果显示两者形成的克隆效率差异无显著性意义，流式细胞术检测结果显示两者有相似的细胞表型，体外诱导分化显示两者都具有成骨、成脂分化能力，但人血小板裂解液扩增的间充质干细胞成骨分化能力更强。提示人血小板裂解液可以取代胎牛血清用于临床低密度扩增人间充质干细胞。

Large-scale expansion of human umbilical cord mesenchymal stem cells in human platelet lysate as a substitute of fetal bovine serum

BACKGROUND：Classic media and fetal bovine serum are commonly used in the culture of umbilical cord mesenchymal stem cells, but the potential risk of serum culture limits its clinical application. OBJECTIVE：To use human platelet lysate alternative to fetal bovine serum for large-scale production of mesenchymal stem cells for therapeutic applications. METHODS：Human platelet lysate was prepared by repeated freezing and thawing, centrifugation, filtration, and concentration. Human umbilical cord mesenchymal stem cells were cultured in Iscove’s Modified Dulbecco’s Medium containing 5%concentrated platelet lysate （experimental group） or 10%fetal bovine serum （control group）. After separation and enzymatic digestion, human umbilical cord mesenchymal stem cells were subcultured at a concentration of 3 000/cm2 up to the fifth generation. Then, cellmorphology and diameter, immune phenotype, osteogenic and adipogenic differentiation, and cloning efficiency were detected and compared between two groups. RESULTS AND CONCLUSION：Human umbilical cord mesenchymal stem cells cultivated in human platelet lysate-supplemented media showed a smal er size and more elongated morphology than those in fetal bovine serum-supplemented media. Colony forming unit-fibroblast analyses further showed no significant differences in colony efficiency. Human umbilical cord mesenchymal stem cells cultivated in human platelet＆amp;nbsp;lysate-supplemented media showed an increase of proliferation capacity;whereas, similar immunophenotypes remained in the two groups. In vitro assays revealed intact differentiation potential. Moreover, human umbilical cord mesenchymal stem cells cultivated in human platelet lysate-supplemented media showed a significantly higher capacity to differentiate towards osteocytes, indicating human platelet lysate is an alternative to fetal bovine serum for low-density production of mesenchymal stem cells for therapeutic applications.