人脐带间充质干细胞原代培养方法的改良

Improved primary cultivation of human umbilical cord mesenchymal stem cells

目的:探讨体外组织块培养原代人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)的改良方法,并观察其形态,免疫表型,成脂成骨分化等生物学特性。方法:从健康足月儿获得脐带,将血管剥离后,余下的结缔组织剪成小块,分别用传统植块法和改良植块法分离培养原代hUC-MSCs。MTT法检测细胞增殖情况并绘制细胞生长曲线,流式细胞仪检测细胞免疫表型,成脂成骨诱导分化检测其分化潜能,化学染色鉴定诱导分化结果。结果:改良植块法脐带组织贴壁培养3~4 d即可从组织块边缘长出长梭形成纤维样原代细胞,5~6 d后细胞可长满80%,比传统植块法原代细胞培养周期缩短了一半以上时间,细胞传代后流水状或漩涡状生长,MTT法显示细胞增殖能力强,流式细胞仪检测提示CD90,CD105,CD73,CD166,CD29,CD44均阳性表达,阳性率均在95%以上,CD45和HLA-DR均阴性表达,阳性率低于2%;诱导分化实验及化学染色结果证实,该细胞具有成脂和成骨多向分化潜能。结论:应用与酶消化法相结合的改良植块法可以获得hUC-MSCs,比传统植块法缩短了原代培养周期,提高了细胞培养效率,所获得细胞贴壁生长,增殖能力强,表达间充质干细胞相关免疫表型,具有多向分化潜能,可在科研和临床应用中作为种子细胞来源。

Objective:To improve the tissue mass primary culture protocol of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs),so as to explore their morphology,cell surface markers,osteogenic and adipogenic differentiation potential and observe biological properties.Methods:Human umbilical cords were obtained from healthy full-term delivered newborns.After stripping off blood vessels,the remaining connective tissues were cut into small fragments,and primary hUC-MSCs were separated by culture of traditional and improved tissue explants methods.The hUC-MSCs were tested with MTT method and the growth curves of them were drawn.Cell surface markers were detected by flow cytometry.After osteogenic and adipogenic differentiation,the potentiality of their differentiation was detected by chemistry staining.Results:After 3-4 days of incubation,flat or spindle-shaped fibroblast-like primary cells were presented,and cells reached 80％ confluence after 5-6 days of incubation.Culture period was shortened by more than half.Flowing water-like or spiral growth morphology was presented after cell passage.MTT revealed high capability of proliferation.Flow cytometry analysis showed CD90 (+),CD105 (+),CD73 (+),CD166(+),CD44(+),CD29(+),positive rate of 95％ or more ;CD45(-),HLA-DR(-),negative rate of not more than 2％.Its differentiation potential was proved by osteogenic and adipogenic differentiation,which was proved by chemistry staining.Conclusion:An improved primary culture method of hUCMSCs has been developed.Primary cells can be obtained from improved tissue explants method combined enzyme digestion technique.Primary cell culture time was shortened,and the efficiency of cell culture was improved.Cells isolated by the new method from the human umbilical cords had the following biological characteristics:adherent growth,high proliferation,mesenchymal stem cell phenotype,and multipotent differentiation.They could be used as the source of seed cells in scientific research and clinical applications.