脐带血有核细胞不同提取和保存方法的比较

Study of isolation,freeze,thaw and storage of nuclear cells from cord blood

目的:通过对脐带血有核细胞(CBNC)的提取和保存方法探讨,为脐带血有核细胞分离、储存和应用提供科学依据。方法:采用3种方法(Ficoll-Paque分离液法、1%甲基纤维素法、红细胞裂解法)分离CBNC,并应用不同储存液在不同时间对细胞进行冷冻和复苏培养。结果:红细胞裂解液法分离所获得的有核细胞数分别是Ficoll-Paque分离液方法的2.9倍(P<0.05)和1%甲基纤维素分离方法的10倍(P<0.01);且红细胞裂解液法所得有核细胞种类(包括淋巴细胞、中性粒细胞和单核细胞)也明显增多;细胞集落形成率与前两种方法比较亦有显著性差异。H3-TdR掺入的细胞活力观察发现,在4℃和20℃条件下存放24h,不同温度下细胞活力没有明显改变。以人脐血血清+DMSO为保护液在液氮储存CBNC 1年,复苏后仍有很强的细胞集落形成能力。结论:应用红细胞裂解液分离提取CBNC获得的细胞数量和种类优于其他两种方法。CBNC冻存时适当加入人脐血清,复苏后克隆形成能力明显增强。

Objective： To study the methods how to isolate and store cord blood nucleated cell（CBNC）. Methods：Using three kind of methods （lymphocyte separating medium,methylcellulose,erythrocyte lysate） to isolate CBNC from cord blood, then freezing, storage and resuscitation with different storage liquid. Results ： The method of erythroeyte lysate could collect far more nucleated cell than other two methods（P〈0. 05 and P〈0. 001） comparing with methods of lymphoeytes separating medium and methylcellulose respectively that including lymphocyte,neutrophilic granulocyte and monocyte. In cell storage study,no difference of nucleated cells activity was found in HS-TdR during 24 hours between 4℃ and 20℃. It could be resuscitation and cultivation forming colony obviously even after one year store in liquid nitrogen canister with human serum from cord blood. Conclusion： Applying erythrocyte lysate to isolate karyoeyte from cord blood and storage with human serum from cord blood is good way for CBNC collection, freezing and storage.