脐带血造血干细胞的分离和冷冻保存研究

Separation and cryopreservation of hematopoietic stem cells from umbilical cord blood units

目的探讨为临床移植有效地分离和冷冻保存无关供者脐带血中的造血干细胞的方法。方法将由普通或改进的两种不同采集袋采集的脐带血,用两步离心法分离、浓缩脐带血中的有核细胞,用BioArchive System(生物档案系统)或常规的程控降温系统将之冷冻,保存在-196℃液氮中。用流式细胞术和细胞培养法检测脐带血中的CD34+细胞和造血祖细胞含量。结果脐带血用改进的采集袋分离后,有核细胞和红细胞的回收率分别为86.4%±4.6%和44.2%±9.6%,而普通采集袋的回收率为78.4%±7.9%和49.8%±11.7%;将富含有核细胞悬液的体积浓缩到20 ml时,有核细胞和红细胞的回收率分别为74.62±9.3%和34.9%±19.1%,浓缩体积为32 ml回收率为86.4%±4.6%和45.1%±7.8%/但两者的CD34+细胞和造血祖细胞的回收率无统计学意义;冷冻脐带血融化后有核细胞、CD34+细胞和CFU-C回收率分别为84.2%±10.1%、96.0%±21.8%和115.1%±23.3%。苔盼蓝拒染率93.8%±4.4%。结论改进的采集袋能够明显提高有核细胞回收率。采用两步离心法和两种冻存方法能够有效地富集和冻存脐带血中的造血干细胞。

Objective To study the efficient approach of separation and cryopreservation of hematopoietic cells from cord blood units. Methods Umbilical cord blood （UCB） units were collected with two different collection bags. Nucleated cells（NC） in UCB were isolated and enriched with two step centrifugation and frozen with BioArchive System or routine programming controlled freezer, then stored in -196℃ liquid nitrogen. The CD34 positive cells were measured with flow cytometry （FCM）, and hematopoietic progenitor cells were assayed with cell culture. Results Isolation with advanced collection bags, the recovery rates of NC and red blood cells（RBC） in UCB were 86.4 %±4.6 % and 44.2 %±9.6 %, respectively,and 78.4 %±7.9 % and 49.8 %±11.7 % respectively in isolation with common bags. When NCs were concentrated to 20 ml, the recovery rates of NC and RBC were 74.62 %±9.3 % and 34.9 % ±19.1% ,and 86.4 %±4.6 % and 45.1%±7.8 % while concentrated to 32 ml. However,the recovery rates of CD34^＋ cells and CFU-C had changed little. After thawed, the recovery rates of NC ,CD34^＋cells, CFU-C and the NC viability were 84.2 %±10.1% ,96.0 %±21.8 %, 115.1%±23.3 % and 93.8 %±4.4 % ,respectively. Conclusion Recovery rate of nucleated cells can be obviously increased by using advanced collection bags. Moreover, hematopoitic cells in the UCB were effectively enriched by two step centrifugation and stored with two cryopreserved methods.