免疫磁珠分离及流式细胞仪分选纯化外周血CD34^+/CD90^+干细胞

Purification of CD34^+/CD90^+ progenitor stem cells using immuno-magnetic microbeads selection and flow-cytometric sorting in peripheral blood leukapheresis

目的 建立人外周血CD34+ 细胞及其亚群的纯化分离方法。方法 用干细胞采集仪收集了 3例病人的外周血干细胞 ,应用免疫磁珠分离柱快速分离其中的CD34+ 细胞 ,随后采用分选型EPICSElite流式细胞仪进一步分选出CD34+ /CD90 + 双阳性早期造血干细胞。结果 免疫磁珠分离纯化后CD34+ 干细胞的纯度可达 83%～ 95 % ,回收率 5 4 %～ 71% ,活细胞率 >95 %。流式细胞仪分选后的CD34+ /CD90 + 细胞纯度可达 90 %以上 ,回收率在 4 0 %～ 5 0 % ,生存率 >95 %。起始标本中CD34+ 细胞含量越高 ,纯化所得到的干细胞纯度和回收率越高。两种纯化后的干细胞在形态学上有明显的不同。结论 联合应用免疫磁珠分离和流式细胞仪分选 ,可以较高的回收率快速纯化高纯度的CD34+

Objective To set up a practical method for purification of CD34 + stem cells and its subsets.Methods Aliquots of leukapheresis samples taken from 3 patients were firstly positive-selected for CD34 + stem cells by immuno-magnetic mircobeads,and then were purified for CD34 +/CD90 + progenitor stem cells using EPICS Elite fluorescence-activated cell sorter.Results The purity of selected CD34+ stem cells by immuno-magnetic separation was 83%-95% with the recovery rate of 54%-71% as well as the viability of more than 95%.After flow cytometric sorting,the purity and viability of CD34 +/CD90 + progenitor stem cell were >90% and >95% respectively,with a recovery of 40%-50%.The higher numbers of CD34 + stem cells in the beginning apheresis have,the higher purity and recovery of the procedures have.The morphologic characteristics between the two separations were different.Conclusions We established an effective procedure for purification of CD34 + stem cells and its subsets using combination of immuno-magnetic mircobeads selection and fluorescence-activated flow cytometric sorting,with satisfied cell purity,viability and recovery.