人外周血造血干细胞低温保存方法的实验研究

Study on cryopreservation of human peripheral blood stem cells

目的 观察 -196℃程控降温冻存方法和 -80℃直接冻存方法中单用二甲基亚砜 (DMSO)与DMSO加羟乙基淀粉 (HES)两种冻存保护剂对人外周血造血干细胞体外冻存的效果。方法 对5例经Cs-3000Plus血细胞分离机分离所得的外周血单个核细胞分别加入10 %DMSO和5 %DMSO加6%HES冻存保护剂。并分别以 -196℃程控降温冻存方法和 -80℃直接冻存方法保存15d ,以快速复温法复苏细胞 ,检测冻存前后细胞活率及有核细胞、CD34+细胞、粒单系集落形成单位 (CFU -GM)、红系集落形成单位 (CFU -E)回收率。 结果 在 -196℃程控降温冻存方法时 ,单用DMSO和DMSO加HES两组在细胞活率、有核细胞回收率、CD34+细胞回收率、CFU -E回收率方面差别无显著性意义 ;在 -80℃直接冻存方法时 ,MDSO加HES组有核细胞回收率、CFU -E回收率高于单用DMSO组。结论 -196℃程控降温冻存时10%DMSO和5 %DMSO加6 %HES两种冻存保护剂均可有效应用于外周血造血干细胞保存 ;-80℃直接冻存方法适合5 %DMSO加6%HES冻存保护剂 ,也可有效用于外周血造血干细胞保存。

Objective To compare different methods in human stem cell conservation.Method Five PBMNCs samples extracted by Cs_3000 blood cell separator from 5 patients were cryopreserved in a cryoprotectant solution composed of 10% dimethylsulfoxide(DMSO) or 5% DMSO supplemented with 6% hydroxyethyl starch(HES) alternatively.Each samples were conserved by both -196℃ program_controlled freezing procedure or -80℃ direct freezing procedure for 15 days and were then recovered by 40℃water bath.The pre_and post_freezing cell livability were compared and the recovery rate of nuclear cells,CD34+ cells,CFU_GM and CFU_E were evaluated.Results No significant differences in cell livability and the recovery rate of nuclear cells,CD34+ cells and CFU_E were found in two freezing systems under the -196℃ program_controlled freezing procedure.However,an increase of nuclear cells and CFU_E recovery rate were detected for 5% DMSO plus 6% HES in the -80℃ direct freezing procedure.Conclusion Both two cryoprotectant solutions can be effectively applied in peripheral blood cell conservation for -196℃ program_controlled freezing procedure;-80℃direct freezing procedure can also be effectively applied in peripheral blood cell conservation with the cryoprotectant contains 5% DMSO plus 6% HES.