程控降温法与二步法对胎盘间充质干细胞冻存效果的影响

Effects of cryopreservation methods on human pMSCs: programmed versus stepwise freezing

目的观察对比程控降温法与二步法对胎盘间充质干细胞深低温冻存效果的影响。方法分别采用程控降温法和二步法对3个批次的pMSCs进行液氮冻存,3个月后进行复苏,测定细胞存活率、活细胞数,观察体外培养1~4 d时的细胞形态,CCK8法进行细胞增殖检测,同时用流式细胞术鉴定细胞表面标记抗原,用油红O染色、茜素红S染色和Masson染色进行细胞三向分化能力鉴定。结果程控降温法对比二步法冻存的细胞活率在冻存前、复苏后均无显著差异(P>0.05)。倒置显微镜观察细胞形态,均在24 h时贴壁,呈短梭形,96 h时细胞密度达到95%左右,均匀铺满视野。增殖测定结果显示,培养第3~5天为两组细胞的对数生长期,第5天时增殖倍数达到最高,分别为第1天的6.44倍和6.29倍,经过短暂的平台期后进入衰亡期,增殖趋势一致;但分别在第4天(P<0.01)、第6天(P<0.05)和第7天(P<0.05)时,程控降温法冻存的细胞存活率极显著或显著高于二步法冻存的细胞。两种方法冻存细胞的表面标志性抗原CD73、CD90、CD105阳性细胞数百分比分别为99.85%、99.87%、96.45%和99.73%、99.73%、96.17%。诱导14 d后两组pMSCs均出现明显的成脂、成软骨、成骨细胞特性。结论采用程控降温法或二步法冻存pMSCs均能很好地维持细胞活力及生物特性,在条件允许情况下宜采用程控降温法,以获得增殖能力更好的种子细胞。

Objective To evaluate the effects of programmed or stepwise freezing on human placental mesenchymal stem cells（pMSCs）. Methods Programmed or stepwise freezing was applied to three batches of pMSCs cryopreservation at-196 ℃ for three months. Cell viability and proliferation were measured through Trypan blue staining and CCK8 assay after being thawed and seeded, respectively. We further determined the characteristic antigenic markers of mesenchymal stem cells by flow cytometry. The adipogenic, osteogenic and chondrogenic differentiation capacities of pMSCs were also tested by Oil red O, Alizarin red S and Masson staining, respectively. Results There was no significant difference with respect to the cell viability between programmed and stepwise freezing of pMSCs before or after being thawed（P〈0.05）. Both groups of pMSCs adhered after 24 h culture, and reached about 95% density after 96 h. The pMSCs proliferation was in a logarithmic phase between 3 to 5 d cell culture and then reached to a declined phase. Cell viability of pMSCs cryopreserved by programmed freezing was significant higher than that by stepwise freezing at 4 d（P〈0.01）, 6 d（P〈0.05） and 7 d（P〈0.05） culture. Moreover, both methods preserved the expression of antigenic markers and differentiating capacity of pMSCs. Conclusion The cell viability and biological characteristics of pMSCs can be maintained well by programmed method or stepwise cryopreservation. It is worth mentioning that the programmed cryopreservation method should be used to obtain the seed cells with better proliferation ability under permissible conditions.