微载体cytodex 3大量扩增成人骨髓间充质干细胞的初步研究

Scale-Up Culture of Adult Human Mesenchymal Stem Cells on Microcarrier Cytodex 3

目的建立一种体外分离、大量扩增培养成人骨髓间充质干细胞(hMSCs)的方法。方法用Percoll梯度离心结合贴壁法分离hMSCs,用微载体cytodex 3培养hMSCs,以普通单层聚苯乙烯(TCPS)培养作对照,用流式细胞仪(FCM)和MTT法对其细胞表型和增殖活性检测。结果①梯度离心结合早期换液是分离hMSCs的较好方法,FCM检测表明hMSCs表面表达CD29、CD44和CD105,而不表达CD14、CD34、CD45、VLA-1和HLA-DR。hMSCs细胞周期分布显示:G0/G1期(86.4±3.8)%,S+G2+M期(13.6±4.2)%;②hMSCs与cytodex 3有良好的相容性,MTT法表明hMSCs在cytodex 3表面悬浮生长时具有比普通单层TCPS培养时更高的数量和增殖活性,FCM分析表明两者的细胞表型和细胞周期分布相同,无显著性差异(P>0.05)。结论微载体cytodex 3培养方法是扩增组织工程种子细胞hMSCs的有效方法。

Objective To establish a method for the isolation and scale-up culture of adult human bone marrow mesenchymal stem cells（hMSCs） on microcarrier cytodex 3. Methods The density gradient centrifugation separation combining with adherent plating with early changed culture medium was applied to separate hMSCs. The phenotype, cell cycle and proliferation of the hMSCs cultured on microcarrier cytodex 3 and tissue culture polystyrene （TCPS） were detected by FCM and MTT assay. Results The density gradient centrifugation separation combining with adherent plating with early changed culture medium was an economical and efficient method to separate hMSCs. The hMSCs express CD29, CD44 and CD105, whereas the expression of CDI4, CD34, CD45, VLA-1 and HLA-DR was not detected. The cell cycle of the hMSCs cultured on TCPS showed that the ratio of G0/G1 phase was （86.4± 3.8） % , and the ratio of S ＋ G2 ＋ M was （13.6 ±4.2）%. The amount and proliferation of hMSCs cultured on cytodex 3 was higher than those cultured on TCPS, but cell cycle and phenotype were not significantly different between the hMSCs cultured on cytodex 3 and TCPS （P 〉 0.05）. Conclusion Microcarrier cytodex 3 culture system is a suitable way to expand the hMSCs for tissue engineering.