人羊水间充质干细胞分离培养方法的优化

Optimization of the isolation and cultivation method of human amniotic fluid-derived mesenchymal stem cells

背景:目前关于人羊水间充质干细胞的体外分离培养方法不一,获得较多数量的间充质干细胞仍有困难。目的:分离80份羊水标本,比较不同的体外培养方法,优化筛选到一种合适的培养纯化条件,为人羊水间充质干细胞的基础研究和临床广泛应用奠定基础。方法:无菌条件下采集足月分娩和终止妊娠引产的羊水,分离羊水细胞,比较孕期、不同培养基、接种密度、首次换液时间对人羊水间充质干细胞原代培养过程的影响,通过细胞化学染色方法检测细胞代谢情况,观察人羊水间充质干细胞的生物学特性。结果与结论:相同培养条件下,采用AmnioMAXⅡcomplete羊水专用细胞培养基,以5×104/cm2的密度接种,首次换液时间为6d时,人羊水间充质干细胞原代培养成功率较高。孕期为14～20周的人羊水间充质干细胞培养成功率高于晚期妊娠(21～36周)及足月分娩(37～42周)的羊水。人羊水间充质干细胞强表达过碘酸-雪夫、酸性磷酸酶、酸性α-醋酸萘酚酯酶染色,弱表达中性粒细胞碱性磷酸酶,不表达过氧化酶、苏丹黑染色。提示,优化筛选到一种合适的人羊水间充质干细胞培养纯化条件。

BACKGROUND：There are different methods of in vitro isolating culture for human amniotic fluid-derived mesenchymal stem cells（AF-MSCs）.To gain most AF-MSCs is still difficult.OBJECTIVE：We had isolated 80 cases AF,and compared different in vitro culture methods for optimization and bolted a better method to establish foundation of AF-MSCs in clinical widespread application.METHODS：Amniotic fluid was collected from full-term deliveries and artificial labors under aseptic condition.The amniotic fluid cells were isolated by centrifugation.The influences of different pregnancy ages,culture media,incubation densities and the time points of the first medium change on the growth of AF-MSCs were analyzed.The cell metabolism was determined and biological characteristics of AF-MSCs were observed by cytochemical staining.RESULTS AND CONCLUSION：When the other conditions were the same,AmnioMAXⅡcomplete medium was the best medium.5×104/cm2 was the best planting density;the best time for the first medium change was on the sixth day.The achievement ratio of amniotic fluid from fetation of intermediate stage（14-20 weeks） was higher than that of later fetation（21-36 weeks） and full-term deliveries（37-42 weeks） when the other conditions were the same.AF-MSCs highly expressed periodic acid-schiff,acid phosphatase,α-naphthol acetate esterase（α-NAE） and weakly expressed neutrophil alkaline phosphatase,but no peroxidase and Sudan black was found.Results suggest that we have found the optimized method of culturing AF-MSCs.