脐带血造血干细胞分离冻存方法优化

Optimization of Cord Blood Hematopoietic Cells Isolation and Cryopreservation Method

探讨了不同分离方法和冻存方法对脐带血造血干细胞的分离和保存效果的差异。对比了离心沉降法和自然沉降法分离效果,以及DMSO和氯化钠混合液,DMSO和右旋糖酐混合液,DMSO和羟乙基淀粉混合液,DMSO、胎牛血清和F12培养基混合液等4种冷冻保护剂效果,并对冻存后细胞复苏进行有核细胞计数、台盼蓝染色、流式细胞术、细胞集落培养等检测。通过检测结果筛选了最佳冻存方法,同时研究了冻存时间对脐带血造血干细胞的影响。结果表明:离心沉降法效果最好且效率高;DMSO和羟乙基淀粉混合液冻存效果最佳且成本更低;脐带血造血干细胞冻存12个月内细胞各方面差异不明显。

In order to show the effects of different methods of cord blood hematopoietic cells isolation and preservation, the differences of density gradient centrifugation method and natural sedimentation method were compared. We also compared the low temperature protective effect of mixed solution of sodium chloride and dimethyl suifoxide （DMSO） , the mixed solution of DMSO and dextran, the mixed solution of DMSO and hydroxyethyl starch, the mixed solution of DMSO and Fetal Bovine Serum and F12 medium and so on. Post-thawed cells were detected by means of trypan blue dye exclusion test, flow cytometry analysis, ceils colony assay to find the best cell freezing medium and to study the effect of cord blood hematopoietic cells cryopreservation for different time. The results show that density gra- dient centrifugation is the best isolation method. The best freezing medium of umbilical cord blood stem cells is the mixed solution of DMSO and hydroxyethyl starch. There is no obvious difference a- mong the umbilical cord blood stem cells cryopreservation for different time within 12 months.