不同孕期羊水间充质干细胞体外培养方法的比较

背景：以往对羊水间充质干细胞的培养多采集孕中期羊水,对妊娠其他阶段羊水进行间充质干细胞培养的研究还很少,特别是系统地对不同孕期羊水和不同培养基进行间充质干细胞培养的效果还未见报道。目的：比较不同孕期羊水中间充质干细胞体外培养的效果。方法：采用两种不同细胞培养基分别对60份妊娠时间为15~42周的羊水进行间充质干细胞的分离培养,观察间充质干细胞培养过程中生长状况及间充质干细胞培养成功率;细胞化学染色法检测间充质干细胞的化学性质;MTT法检测传代后间充质干细胞的生长曲线。结果与结论：采用含体积分数为10%胎牛血清的PRMI-1640培养基和AmnioMAXⅡ complete羊水专用细胞培养基对孕期为37~42周羊水的间充质干细胞培养成功率分别为8%和44%,孕期为21~36周羊水的间充质干细胞成功率分别为20%和60%。而孕期为15~20周的羊水用两种培养基培养成功率为100%。细胞化学染色结果显示：POX、SB(-),ACP、PAS、AENE(+),而NAP有1%为(+),不同孕期染色结果无差异。MTT法测定羊水间充质干细胞的生长曲线呈“S”形。孕期在15~36周的羊水间充质干细胞可以传15代以上仍然旺盛,而孕期为37~42周的羊水间充质干细胞传10代即表现为生长缓慢。结果表明采用AmnioMAXⅡ complete羊水专用细胞培养基可以明显提高晚期羊水间充质干细胞培养的成功率。羊水的最佳采集时间为15~20周。

Comparison of in vitro culture of mesenchymal stem cells in the amniotic fluid from different duration of pregnancy

BACKGROUND: Mid-half amniotic fluid was usually collected for mesenchymal stem cells (MSCs) culture, but the study on the other phase amniotic fluid-MSCs was seldom. The culture effect of amniotic fluid from different duration of pregnancy with different media is not reported. OBJECTIVE:To compare the in vitro culture effect of MSCs in the amniotic fluid from different duration of pregnancy. METHODS:Two media were used to isolate and culture MSCs from 60 cases amniotic fluid of pregnancy 15-42 weeks. The growth state and the success rate of MSCs cultivation course were observed. The cytochemical staining was applied to detect the chemical characteristics of MSCs. Growth curve of MSCs after passage was detected by MTT assay. RESULTS AND CONCLUSION:The success rates of 37-42 weeks pregnancy amniotic fluid MSCs were 8% and 44% using PRMI-1640 and AmnioMAXⅡ complete medium containing 10% fetal bovine serum, that of 21-36 weeks were 20% and 60%; but that of 15-20 weeks was 100% using any medium. The result of cytochemical staining showed that POX and SB were negative; PAS, ACP, AENE were positive; NAP was 1% positive. No difference was determined in staining results at different duration of pregnancy. MTT assay showed that growth curve of MSCs exhibited “S” shape. Amniotic fluid-MSCs of 15-36 week pregnancy were transferred for 15 passages, showing prosperity; that of 37-42 weeks could only be transferred for 10 passages, showing slow growth. The results demonstrated that AmnioMAXⅡ complete medium can elevate obviously the success rate of amniotic fluid-MSCs from late-phase amniotic fluid. The optimal collection time of amniotic fluid was 15-20 weeks.