人骨髓间充质干细胞体外分离培养方法的改进

Modified Isolation and Cultivation of Human Mesenchymal Stem Cells in Vitro

目的:对人骨髓间充质干细胞(Mesenchymalstemcells,MSCs)的体外分离纯化、培养扩增方法的改 进,为MSCs的进一步诱导分化和应用奠定基础。方法:抽取人骨髓细胞,采用密度梯度离心结合贴壁培养法 进行分离纯化,并传代扩增,测定生长曲线和贴壁率,形态学观察,流式细胞仪检测其表面抗原表达。结果:密 度梯度离心结合贴壁培养法能有效分离纯化人骨髓MSCs,MSCs在含胎牛血清的L DMEM培养液中生长性状 相对稳定,细胞基本呈成纤维细胞样生长,1、3、5代细胞生长曲线、贴壁率基本相同,贴壁存活率高。表达 CD29、CD44、CD105、CD166。结论:密度梯度离心结合贴壁培养法能有效分离纯化人骨髓MSCs,在含胎牛血清的 L DMEM培养液中,细胞稳定扩增。

Objective:To modify the isolation and cultivation of mesenchymal stem cells(MSCs) from human bone marrow for offering an experimental foundation for their further differentiation and actual application. Methods: Isolated and purified the human marrow MSCs by density gradient centrifugation and culture plastic-adhering. After in vitro subculture and expansion, observed the morphology of the cells, drew the growth curve, measured the adhesive rate, analyzed MSCs by flow cytometry. Results: The living characteristic was stable in L-DMEM medium containing newborn bovine serum. The growth curves and adhesive rates of passage 1, 3, and 5 were quite similar. MSCs exhibited a typical fibroblastlike morphology and expressed CD 29, CD 44, CD 105, CD 166. Conclusion: The marrow MSCs could be effectively isolated and purified by density gradient centrifugation and culture plastic-adhering and expanded satisfactorily in L-DMEM with newborn bovine serum.