低温冻存对人绒毛膜来源的间充质干细胞多向分化潜能的影响

Influence of cryopreservation on multilineage differentiation potential of human chorion-derived mesenchymal stem cells

目的探讨低温保存对人绒毛膜来源的间充质干细胞(hCDMSC)体外生长特性和多向分化潜能的影响,验证冻存后hCDMSC作为组织工程种子细胞的可行性。方法用胶原酶和胰蛋白酶法消化胎盘组织基蜕膜,传代培养,取第4代细胞置于液氮深低温(-196℃)条件下冻存3个月后复苏,采用MTT法测定复苏hCDMSC的增殖活性,茜素红和油红O染色检测其成骨、成脂诱导分化情况。未经低温保存的hCDMSC作为对照。结果低温保存复苏后的hCDMSC增殖活性、多向诱导分化能力与未冻存细胞比较,差异无统计学意义,细胞的茜素红和油红O染色呈阳性反应。结论低温保存对hCDMSC体外生长能力和多向诱导分化能力没有影响,冻存后hCDMSC可以作为组织工程的种子细胞。

Objective To explore the influence of cryopreservation on growth characteristics and multilineage differentiation potential of human chorion-derived mesenchymal stem cells（hCDMSC） in vitro and verify the feasibility of cryopreserved hCDMSC serving as seed cells for tissue engineering.Methods Collagenase and trypsin were used to trypsinize placental basal decidua,cells were subcultured and the fourth generation cells were resuscitated after 3 months cyropreservation in liquid nitrogen under the conditions of deep hypothermia（-196 ℃）.MTT method was employed to measure the cellular proliferation of resuscitated hCDMSC and alizarin red and oil red O staining were applied to detect the osteogenic and adipogenic differentiation.hCDMSC without cyropreservation served as a control.Results There were no significant difference in proliferation activity and multilineage differentiation potential of resuscitated hCDMSC after cryopreservation compared to those without cryopreservation.hCDMSCs demonstrated positive staining with aizarin red and oil red O.Conclusion Cryopreservation has no influence on proliferation and multilineage differentiation potential of hCDMSC in vitro,and cryopreserved hCDMSCs can serve as seeds for tissue engineering.