脐血干细胞玻璃化冻存技术研究

Cryopreservation of umbilical cord blood stem cells by vitrification

以羟乙基淀粉(HES)法分离所得的脐血干细胞作为材料,进行玻璃化超低温保存,探索和建立最合适的保护剂浓度和预处理时间,以期在细胞冻融后获得较高的活率.采用系列体积分数的玻璃化冷冻保护剂(PVS2)预处理不同的时间,样品在完全浓度的玻璃化保护剂中直接存入液氮.冻融后的细胞经过噻唑蓝(MTT)法和FDA PI双荧光染色法检测存活率,获得预处理过程的优化条件:体积分数为60%的PVS2,预处理15min,FDA PI值为46.43.用海藻糖取代原保护剂中的蔗糖,与同样条件下含蔗糖保护剂处理的细胞相比较,FDA PI最大值高于后者20.25%,CD34+回收率最大值高于后者25.64%,表明含海藻糖的保护剂在该冻存技术中的保护效果明显优于含蔗糖的保仿剂.

Umbilical cord blood stem cells separated with hydroxyethyl starch (HES) were cryopreservated by vitrification to establish the optimal cryoprotectant concentration and pretreatment time for obtaining higher survival rate of recovered cells. Samples were disposed to a series concentration of cryoprotectant (PVS2) for different length of time and equilibrated in complete strength of the vitrification solution before the cryotubes containing treated cells and cryoprotectant were plunged directly into liquid nitrogen. The survival rate of the recovered cells was tested by MTT and FDA-PI. The preferable vitrification conditions were found to be: the cryoprotectant concentration of 60% and disposal time of 15 min. Under these conditions, FDA-PI was 46.43. Trehalose was selected for substituting sucrose in the cryoprotectant. The vitrification process was under the same preferable conditions. The survival rate of the recovered cells was tested by FDA-PI and CD34+ cell-count. For FDA-PI, trehalose was 20.25% higher than sucrose; and for CD34+ cell-count, trehalose was 25.64% higher than sucrose. It shows that trehalose is superior to sucrose during cryopreservation.