-80℃条件下不同细胞浓度外周血干细胞冻存效果的实验观察

The observation of the effect of different cell concentration on peripheral blood stem cells (PBSC) cryopreservation under -80℃

目的 观察不同细胞浓度外周血干细胞在 - 80℃条件下的冻存效果。方法 采用一种简便的 - 80℃干细胞冻存方法 ,其干细胞保护剂终浓度为 5 %二甲基亚砜 (DMSO) ,3%羟乙基淀粉 (HES)和 4 %人血白蛋白 (HSA) ,不经程序降温 ,直接 -80℃冰箱冻存外周血干细胞 ,并在不同时相点 (冻存后 1、3、6、9、1 2个月 )对推荐细胞浓度组 (1 .3～ 3.9)× 1 0 7/ml、调整细胞浓度组 (8.0～ 2 9.9)× 1 0 7/ml和高细胞浓度组 (30 .1～ 70 .1 )× 1 0 7/ml的外周血干细胞的细胞活性进行检测 ,观察台盼蓝拒染率和MNC、CFU GM、CD34+ 细胞的回收率。结果 3组干细胞活性在 1 2个月内无明显下降 ;干细胞冻存后 9个月期间 ,3组冻存细胞的台盼蓝拒染率 ,MNC回收率、CFU GM回收率、CD34+ 细胞回收率都在 80 %以上。冻存 1 2个月中 ,3组在冻存后同一时间相互比较无统计学差异 (P >0 .0 5 ) ,说明该 - 80℃干细胞冻存方法适用于不同浓度的细胞液 ,短期内具有良好的冻存效果。结论 5 %DMSO、3%HES和 4 %HAS作为干细胞冷冻保护剂直接 - 80℃冰箱冻存外周血干细胞的方法能有效地保护不同浓度细胞液的外周血干细胞活性 ,适当地提高细胞保存时的浓度 ,减少回输量 。

Objective To observe the effect of cell concentration on peripheral blood stem cells (PBSC) cryopreservation under -80℃. Methods Under -80℃ with protective agent which final density was 5%DMSO, 3%HES, 4%HAS, PBSC were cryopreserved at cell concentration recommended (1.3 3.9)×10 7/ml),cell concentration adjusted (8.0 29.9)×10 7/ml) and high cell concentration (30.1 70.1)×10 7/ml).After thawing, the cell viability including the number of MNC and the recovery of CFU GM and CD34 + cells were measured at the different time (1,3,6,9,12 months after cryopreservation). Results Cell viability had no significant decline within 12months in each group. The recovery of MNC,CFU GM and CD34 + cells at cell concentration recommended, cell concentration adjusted and high cell concentration was above 80% respectively in 9 months, which were no significantly decreased( P >0.05), compared with those in 1 month; There was no significant difference in the recovery of MNC,CFU GM and CD34 + cells among cell concentration recommended , cell concentration adjusted and high cell concentration of PBSC cryopreserved at the same time. It is enough to make sure that the PBSC transplantation be successful by cryopreservation of PBSC in -80℃ freezer with different cell concentration. Conclusion It was suggested that, the cryopreservation method of PBSC in -80℃ freezer with 5%DMSO, 3%HES, 4%HSA as its protective agent be effective to the different cell concentration. The concentration of cell suspension to be cryopreserved may be raised appropriately and the total volume decreased properly in order to alleviate the clinical side reaction.