脐血造血细胞的冷冻保存方法的比较

Comparison of cryopreservation methods of umbilical hematopoietic stem/progenitor cells

目的 :比较程控降温和 - 70℃冰箱直接保存脐血造血细胞对细胞活性的影响及免疫磁珠分离法对细胞生物学活性的影响。方法 :用密度梯度离心法分离出脐血中单个核细胞后 ,采用联合冷冻保护剂经程控降温后存放于液氮和直接存放于 - 70℃冰箱两种方法冷冻保存一个月 ,从细胞活力、细胞回收率、集落形成能力、集落回收率等方面比较这两种冷冻方法的优劣。另外 ,用免疫磁珠分离法正性分选脐带血中CD34+细胞 ,流式细胞仪计数分选前后的CD34+细胞的纯度及回收率 ,并分别进行分选前后的细胞培养。结果 :细胞活力、细胞回收率、集落形成能力、集落回收率在冷冻的两组间差异不显著。用免疫磁珠分选后CD34+细胞纯度达 88.3% ,回收率为 4 6 .2 % ,分选后的CD34+细胞比分选后CD34 细胞的细胞活力及集落形成能力明显要高。结论 :两种方法均可对脐血造血细胞进行保存。免疫磁珠分选法能够提供高纯度的造血干细胞 ,其分选过程不会影响细胞的活力及集落形成能力 。

Objective To compare the different effects of the programmable cell freezer and the -70℃ mechanical freezer in the preservation of the combilical cord blood and to study the effects of magnetic activated cell sorting on cell biologic characters. Methods After separating the nucleated cells from the umbilical blood by the density gradient separation method, we used cryoprotectant to protect the nucleated cells and preserve them by two methods; one was using the programmable cell freezer to cool the cells and finally putting them to the liquid nitrogen, and the other one was directly preserving the cells in a -70℃ mechanical freezer. The cells were thawed after a month and tested for the viability and clonogenic ability. Cell recovery, clonogenic capacity and clonogenic recovery by the two cryopreservation methods were compared. Meanwhile, the CD34+ cells were sorted by the method of magnetic cell sorting from the umbilical cord blood, and the cell purity and recovery after sorting were analyzed by the method of fluorescence activated cell sorting. Results There were no significant differences between the two preserving methods in cell viability, cell recovery, clonogenic capacity, or colony recovery. The purity of the CD34+ cells was 88.3% and the recovery rate was 46.2% after the cell sorting. The cell viability and cologenic capacity of the CD34+ cells were much higher than those of the CD34- cells. Conclusion The umbilical cord blood can be efficiently preserved by the two methods and highly purified CD34+ cells could be obtained by using the method of magnetic cell sorting. The procedure of cell sorting doesn't affect the viability and clonogenic ability. The study provides some evidence for clinical application of preserving the umbilical cord blood and transplanting CD34+ cells.