-80℃非程控冷冻保存人外周血干细胞的效果观察

Cryopreservation of human peripheral blood stem cells by -80℃ non rate-controlled freezing method

目的探讨-80℃非程控冷冻保存人外周血干细胞（PBSC）的效果。方法41份PBSC标本，39份采自恶性肿瘤患者，2份采自正常供者。对于恶性肿瘤患者，干细胞采集前采用针对性化疗方案联合粒细胞集落刺激因子或粒一单细胞集落刺激因子进行动员。将采集的干细胞与采用120g／L羟乙基淀粉、体积分数为10％二甲基亚砜及80g／L人血清白蛋白组成的冷冻保护剂按1：1混合，然后将其直接放人-80℃低温冰箱内保存。测定保存前以及保存后不同时间的单个核细胞（MNC）、有核细胞（NC）、CD34＋细胞、粒一单核细胞系集落形成单位（CFU-GM）和红系爆式集落形成单位（BFU-E）等的回收率以及细胞的台盼蓝拒染率。结果经-80℃非程控直接冷冻保存1个月至10年，PBSC的台盼蓝拒染率、NC和MNC回收率的变化较小（P〉0．05），而CD34＋细胞、CFU-GM和BFU-E回收率在冻存5～10年后下降较明显（P〈0．05），但仍分别达89．6％、85．1％和83．7％。34例患者经相应处理后，回输经-80℃下保存13-53d（平均19d）的干细胞，患者于细胞回输后11～27d（平均14．7d）获得造血功能重建。结论在采用终浓度为60g/L羟乙基淀粉、体积分数为5％二甲基亚砜及40g／L人血清白蛋白组成的冷冻保护剂的情况下，-80℃非程控冷冻法适于PBSC的长期保存，效果较理想。

Objective To investigate the cryopreservation of human peripheral blood stem cells （PBSC） by -80℃ non rate-controlled freezing method. Methods Forty-one samples of PBSC were obtained from 39 malignant tumor patients and 2 normal donors. PBSC in all malignant tumor patients were mobilized with special chemotherapy protocols combined with granulocyte colony-stimulating factor or granulocyte/monocyte colony-stimulating factor before collection. The obtained PBSC were mixed with same volume of cryoprotectant composed of 12 g/L hydroxyethyl starch, 10 dimethylsulfoxide and 80 g/L human serum albumin, and then cryopreserved in -80℃ deep freeze refrigerator directly. Trypan blue viability and recovery rate of nucleated cells （NC）, mononuclear cells （MNC）, CD34＋ cells （CD34＋ ）, colony-forming unit-granulocyte macrophages （CFU-GM）, burst forming unit-erythroid （BFU-E） were detected before and after cryopreservation in different periods. Results Having been cryopreserved from 1 month to 10 years in -80℃, there were no statistically significant differences in trypan blue viability, NC and MNC recovery rate （P〈0. 05）. Although recovery rate of CD34＋, CFU-GM and BFU-E was decreased obviously after cryopreservation for 5-10 years （P〈0. 05）, 10-year recovery rate was 89. 6 %,85. 1 % and 83. 7 %, respectively. Thirty-four patients subject to corresponding pretreatment were transfused with -80℃ cryopreserved PBSC which had been stored for 13 to 35 days （mean 19 days） and hematological reconstitution was obtained successfully in 11 to 27 days （mean 14. 7 days）. Conelusion -80℃ non rate-controlled freezing method is suitable to human PBSC long-term cryopreservation with the cryoprotectant composed of 60 g/L hydroxyethyl starch, 5 % dimethylsulfoxide and 40 g/L human serum albumin, and its cryopreservation effect is ideal.