不同培养方法对骨髓间充质干细胞贴壁和纯度的影响

Effect of different culturing methods on the attachment and purity of human Mesenchymal stem cells

目的探讨骨髓间充质干细胞(MSCs)理想的分离、培养方法。方法将MSCs分别于普通培养瓶、Co60培养瓶、胶原培养瓶中培养,观察三批标本细胞贴壁情况;将3个批次标本细胞分别用IMDM、1640和Mesencult培养液培养,观察细胞纯化情况;流式细胞术检测细胞表面标志。结果胶原培养瓶及Co60培养瓶细胞贴壁,情况明显好于普通培养瓶,且后者费用低;MesenCult培养体系能获得高纯度MSCs,且传代后细胞不易分化。流式细胞术示三种培养体系中细胞表型均表现为CD34(-)、CD45(-)、CD71(++)、CD44(++)、CD90(++)。结论应用Co60照射处理的培养瓶结合MesenCult培养基可以收获大量、高纯度的MSCs;本实验为MSCs进一步的研究及其用于临床提供了实验方法及依据。

Objective To develop the better methods of isolating and culturing mesenchymal stem cells（MSCs）. Method The attachment efficiency of MSCs cultured in normal culture flask, flask rayed by Co^60 and Ⅳ collagen coating flask were observed, while the purity of MSCs cultured in IMDM, 1640 and MesenCult medium were deceted.The expression of surface molecules were examined by flow cytometic. Results Culture flask covered by collagen type Ⅳ or rayed by Co^60 （6GY） was better for the adherence and growth of MSCs. The purity of MSCs cultured in mesencult serum was the highest, and the ceils didn＇t differentiate in several passage. Flow cytometric analysis indicated that MSCs were CD34（ - ）, CD45 （ - ）, CD71 （ ＋ ＋ ）, CD44（ ＋ ＋ ）, CD90（ ＋ ＋ ）. Conclusion A great number of MSCs with high purity can be obtained by culturing in flask rayed by Co^60 （6GY） with mesencult serum free media. This study can provide a feasible empirical method for further basic research and clinical application.