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目的体外分离培养人羊水间充质干细胞(human amniotic fluid-derived mesenchymal stem cells,HAFMSCs),观察低温冻存复苏后HAFMSCs生物学特征,为进一步研究奠定理论基础。方法取12份自愿捐赠的孕16～20周羊水标本,采用改良两步法分离培养HAFMSCs,用含量不同的FBS、DMSO冻存液冻存细胞,液氮冻存12周后42℃水浴复苏,锥虫蓝染色检测细胞存活率,MTT法检测细胞增殖速度并绘制生长曲线,流式细胞仪检测冻存复苏后HAFMSCs表型。对冻存复苏后的HAFMSCs进行成脂、成骨诱导分化培养,并分别采用油红O、von Kossa染色进行鉴定;实时荧光定量PCR分析细胞冻存前后Oct-4、Nanog mRNA表达差异。结果细胞冻存12周后,不同的冻存方案对细胞存活率影响有差异,优化的冻存方案为DMEM/FBS/DMSO=50%/40%/10%。冻存复苏后的HAFMSCs呈漩涡状排列,生长曲线呈S形,与冻存前细胞生长曲线相似。流式细胞仪检测示冻存复苏后细胞的MSCs表型CD29、CD44、CD73、CD90为阳性,造血干细胞表型CD34、CD45为阴性。成脂、成骨诱导21 d,油红O、von Kossa染色均呈阳性。实时荧光定量PCR检测示冻存前后Oct-4、Nanog mRNA表达水平差异无统计学意义(P>0.05)。结论 HAFMSCs具有体外增殖快、分化能力强的优势;并可耐受短期冻存,复苏后细胞存活率高,生物学特征及分化潜能未发生明显变化,冻存液DMEM/FBS/DMSO=50%/40%/10%是较好冻存方案。
OBJECTIVE: To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs) in vitro and observe the biological characteristics of HAFMSCs after cryopreservation and resuscitation, so as to lay a theoretical foundation for further study. Methods 12 samples of amniotic fluid from 16 to 20 weeks pregnant women donated voluntarily were collected. HAFMSCs were isolated and cultured by modified two-step method. FBS and DMSO cryopreserved cells with different contents were used to freeze the cells. Cell viability was detected by trypan blue staining, cell proliferation rate was measured by MTT assay, and growth curve was drawn. The phenotype of HAFMSCs was detected by flow cytometry. The cryopreserved HAFMSCs were differentiated into adipocytes and osteoblasts and identified by Oil Red O and von Kossa staining respectively. The expression of Oct-4 and Nanog mRNA was analyzed by real-time fluorescence quantitative PCR before and after cryopreservation. Results After 12 weeks of cryopreservation, different cryopreservation protocols had different effects on cell viability. The optimal cryopreservation protocol was DMEM / FBS / DMSO = 50% / 40% / 10%. After cryopreservation and resuscitation, the HAFMSCs were arranged in a swirling pattern with a S-shaped growth curve similar to the cell growth curve before freezing. Flow cytometry showed that the phenotypes of MSCs were positive for CD29, CD44, CD73 and CD90 after cryopreservation, and the phenotypes of CD34 and CD45 were negative for hematopoietic stem cells. Adipogenic, osteogenic induction 21 d, oil red O, von Kossa staining were positive. Real-time PCR showed that there was no significant difference in the expression levels of Oct-4 and Nanog mRNA before and after cryopreservation (P> 0.05). CONCLUSION: HAFMSCs have the advantages of rapid proliferation and strong differentiation in vitro, and can tolerate short-term cryopreservation. The survival rate of the cells after resuscitation is high without significant changes in biological characteristics and differentiation potential. The concentration of DMEM / FBS / DMSO = 50% / 40% / 10% is a better cryopreservation program.