Objective To optimize the procedure for separation and cryopreservation of total nucleated cells (TNCs) from umbilical cord blood. Method Cord blood units were collected and processed using either a double stem cell extraction method or a traditional method. The recovery rate of TNCs and other relevant factors affecting cryopreservation quality were analyzed. Results The average number of TNCs yielded by the double extraction method and the traditional method were (1.52±0.89)× 109and(1.11±0.35)× 109 respectively. The recovery rate of TNCs using the double extraction method was (92.12± 5.78) ％ and using the traditional method it was(86.94± 7.34) ％ (P＜0.05). The viability of TNCs , which were cooled at either 1℃/min or 5℃/min in a controlled-rate freezer from 4 to -40 degrees was (92.4± 2.3)％ and (84.7±2. 4) ％(P＜0.05). The recovery rates of TNCs that were cooled at 1℃/min or 5℃/min in a controlled rate freezer from -40 to -90℃ degrees were (91.1±1.9)％ and (90.3±2.8)％ (P＞0.05)。 The cells were cooled to either -90℃ or -130℃ using a controlled-rate freezer, then moved to liquid nitrogen, and the recovery rates of TNCs were (91.1±2.1)％ and (92.3±1.9)％ ,respectively(P＞0.05)。 Conclusion The double extraction technique provides a high and consistent recovery of TNCs. The optimal method to cryopreserve cord blood mononuclear cells is to cool the cells at 1℃/min to -40℃ and then at 5℃/min to -90℃ using a controlled-rate freezer.%目的 优化脐带血单个核细胞的分离制备及保存的方法.方法 采集符合要求的足月顺产或剖宫产新生儿脐带血,采用二次回浆法分离制备脐带血有核细胞并与传统离心法比较.对分离制备的脐血有核细胞采用不同的降温速率进行低温保存研究,优化降温速率及程序降温终止温度.结果 采用二次回浆法和传统离心法分离制备脐带血有核细胞,其总数(×109)分别为1.52±0.89和1.11±0.35,回收率(%)分别为92.12±5.78和86.94±7.34,差异有统计学意义(P0.05.在程序降温仪降至-90℃和-130℃后移至液氮,细胞回收率(%)分别为91.1±2.1和92.3±1.9,P>0.05.结论 二次回浆法能提高脐带血制备有核细胞数;脐血干细胞保存优化的方法为4℃～-40℃时采用1℃/min的降温速率,再以5℃/min的降温速率降至-90℃后移入液氮保存.
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