人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏

目的：探讨从人脐带华通氏胶（Wharton’s jelly,WJ）中分离、培养、鉴定间充质干细胞（mesenchymal stemcells,MSCs）及其冻存、复苏的方法。方法：采用植块法分离、培养间充质干细胞,流式细胞仪检测P3代细胞免疫表型,鉴定其向成骨、成脂方向诱导分化的能力;将P1细胞冻存6个月后复苏,鉴定复苏后细胞的特性。结果：植块法容易从人脐带华通氏胶中获得间充质干细胞;组织块贴壁后6 d可见组织块周围细胞爬出,原代培养14~18 d细胞融合70%~80%;P3代细胞强烈表达CD73、CD90、CD105,不表达CD14、CD34、CD45、CD79a和HLA-DR;成骨诱导分化后10 d,可见明显钙结节;成脂诱导14 d,有明显的脂滴出现,油红O染色阳性。冻存再复苏细胞活力达80%,细胞免疫表型及成骨、成脂诱导显示与冻存前细胞呈相同的特性。结论：组织块培养法可从人脐带华通氏胶中分离、培养出纯度较高间充质干细胞,冻存、复苏不改变其特性。

Isolation, culture, identification, frozenness and thaw of mesenchymal stem cells from Wharton's jelly of human umbilical cord

Objective： To explore the approach of isolating,culturing,identifing,frozening and thawing MSCs from Wharton＇s jelly of human umbilical cord.Methods： Human umbilical cord mesenchymal stem cells（hUCMSCs） were separated by culture of tissue adherence.The surface of passage 3 cell surface antigen was measured by flow cytometry.The osteogenic and adipogenic differentiation was tested and evaluated by specific staining methods.Passage 1 cells,frozen 6 months,were thawed,then their biological characteristis were identified.Results： MSCs were easily obtained from Wharton＇s jelly of human umbilical cord via the proposed approach of tissue adherence.After 6 days of incubation,cells were presented.The primary cells grew up to 70%~80% confluence after 14~18 days of culture.Flow cytometry analysis revealed that CD73,CD90 and CD105 were highly expressed on the surface of passages 3 cells,but the expression was negative for CD14,CD34,CD45,CD79a and HLA-DR.These cells showed calcium node after culture induction of osteogenic differentiation 10 days later.Furthermore,liquid vacuoles were detected by oil red O staining after culture induction of adipogenic differentiation 14 days later.The living cells of hUCMSCs were 80% after having been frozen and the thawed cells had the same characteristis as privious.Conclusion： Cells from the human umbilical cords have been isolated by tissue culture method,which have the biological characteristics of MSCs.