保存温度对人脐带间充质干细胞制剂细胞存活率的影响

The effect of preservation temperature on the cell survival rate of human umbilical cord mesenchymal stem cell

目的观察保存温度对人脐带间充质干细胞(hUC-MSCs)制剂中干细胞存活率指标的影响,为干细胞制剂的保存与运输提供合理的温控建议。方法新鲜脐带取自2018年12月至2019年6月于天津华兴医院行剖宫产的健康胎儿,均由产妇自愿捐献并签署知情同意书。分离hUC-MSCs后进行培养并传代及制剂制备,之后在4℃、10℃~15℃、25℃、37℃温度下对细胞存活率进行测定。不同保存温度下各组间细胞存活率的比较采用t检验。结果细胞传代到第5代时,流式鉴定显示CD73、CD105和CD90阳性表达达到95%以上,CD45、CD34、CD14、CD19和人类白细胞抗原DR(HLA-DR)表达率在0~1%。细胞三向分化能力呈阳性。在25℃保存温度下,放置10 h时,细胞活率为86.61%,与保存在4℃、10℃~15℃温度比较,细胞活率下降较快(t=3.065,P<0.01)。在37℃保存温度下,放置4 h时,细胞活率为84.92%,细胞活率下降速度最快(t=5.178,P<0.01)。结论hUC-MSCs制剂的适宜保存温度为4℃~15℃,该温度下的最长保存时间为10 h。

Objective This study was to investigate the effect of storage temperature on the survival rate of human umbilical cord mesenchymal stem cells(hUC-MSCs),and to provide reasonable suggestions of temperature control for the preservation and transportation of hUC-MSCs.Methods The fresh umbilical cord was taken from the healthy fetal in Tianjin Huaxing hospital from December 2018 to June 2019.All of them were donated voluntarily by the maternal and signed the informed consent.hUC-MSCs were successfully isolated,cultured and subcultured to the P5.After the preparation,the cell survival rate was measured at 4℃,10℃-15℃,25℃and 37℃.T test was used to compare the cell survival rate among the groups at different storage temperatures.P<0.05 was considered as significant difference.Results When the cells were cultured to the P5,the flow cytometry showed that CD73,CD105 and CD90 were expressed more than 95%,the expression rate of CD45,CD34,CD14,CD19 and human leucocyte antigen-DR(HLA-DR)were 0%-1%.The ability of osteogenic,chondrogenic and lipogenic differentiation were positive.Compared with 4℃and 10℃-15℃,the cell survival rate decreased rapidly(t=3.065,P<0.01).At 37℃,the cell survival rate was 84.92%at 4 hours,with the fastest decreased of cell survival rate(t=5.178,P<0.01).Conclusion The suitable storage temperature of human umbilical cord mesenchymal stem cell is 4℃-15℃,the maximum storage time at this temperature is 10h.