一种简便的外周血干细胞冷冻保存法

A Simplified Method for Cryopreservation of Peripheral Blood Stem Cells

为了简化传统的细胞冷冻方法 ,即用 10 %二甲基亚砜 (DMSO)作为冷冻保护剂并用程控降温仪 - 196℃液氮保存 ,研究了用 5 %DMSO ,3%羟乙基淀粉 (HES)和 4 %人血白蛋白 (HSA)作为冷冻保护剂 ,而不用程控降温仪直接 - 80℃冰箱冷冻细胞的方法。对比了不同方法冷冻保存外周血干细胞 (PBSCs)的细胞活性、单个核细胞数、CFU GM和CD34+ 细胞回收率。结果发现 ,简便冷冻法可获得更高的单个核细胞 (MNC)和CFU GM回收率 ,无凝集现象 ;并且经长达 2 4个月冷冻保存后仍可获得满意的CFU GM和CD34+ 细胞回收率。 37℃融冻和 2 0℃室温融冻法之间各项指标无明显差别 ,细胞融冻后在室温下暴露于 5 %DMSO中 1小时 ,CFU GM回收率并不降低 ,而细胞活性从 93.2 %降至 84 .5 %。所以 ,这种简便的细胞冷冻保存法简便易行 ,可替代传统的液氮保存法 ,对于没有程控降温仪的单位具有实用价值 ,而且还可减少病人DMSO输入量和毒性。

To simplify the traditional method for cryopreservation of peripheral blood stem cells(PBSCs) at -196℃ with rate-controlled freezing with 10% dimethyl sulfoxide(DMSO), the simplified method was carried out by freezing the cells to -80℃ in low-temperature freezer with the combination of 5% DMSO, 3% hydroxyethyl starch(HES) and 4% human serum albumin(HSA) as cryoprotectant. PBSCs were cryopreserved by different methods. Cell viability and recovery rate of mononuclear cells(MNC), CFU-GM and CD34 + cells were compared. It was observed that the higher MNC and CFU-GM recovery rates were achieved and without agglutination with the simplified method. It was also found with this simplified method, satisfactory recovery rates of CFU-GM and CD34 + cells could be obtained when PBSCs were preserved at -80℃ as long as 24 months. There was no difference observed in parameters of cryopreserved PBSCs thawed at 37℃ and 20℃. After the cells being exposed to 5% DMSO at room temperature for 1 hour, the cell viability decreased from 93.2% to 84.5%, however, the CFU-GM recovery rate was not decreased. It is concluded that the simplified cryopreservation technique is better and simpler than the traditional crypreservation method, will be useful for institutions without rate-controlled freezing facility. Moreover, this method diminishes the amount of DMSO infused into patients, thus decreasing its toxicity.