超低温冰箱转液氮阶梯降温法与传统程序降温法保存造血干细胞的比较

Cryopreservation of hematopoietic stem cells with ladder-style freezing from low temperature refrigerator to liquid nitrogen method versus programmed cooling method

背景:在造血干细胞整个冷冻保存过程中,受到降温速率、储存温度深浅、冷冻保护剂组合等因素影响,各国学者在提倡选择何种保存方法上面存在不同的主张。目的:比较-80℃低温冰箱转液氮阶梯降温法与传统程序降温法保存外周造血干细胞的效果。方法:将采集的造血干细胞分两组,第一组细胞浓度为1×1011L-1,加入10%二甲基亚砜,放入程序冷冻仪内程序设置为室温至-4℃按1℃/min的速率降温,按35℃/min快速降至-45℃,再以15℃/min升至-21℃,然后以5℃/min降至-90℃,取出冷冻管置-196℃液氮罐内。第二组细胞浓度为1×1011L-1,加入5%二甲基亚砜、3%羟乙基淀粉、4%人血白蛋白,将冷冻管置-80℃冰箱过夜后取出,置-196℃液氮罐内的气相,再过夜后置液氮罐液相内。结果与结论:两组冷冻方法保存细胞的锥虫蓝拒染率、回收率、凋亡率和死亡率差异无显著性意义(P>0.05)。结果显示用5%二甲基亚砜、4%白蛋白、3%羟乙基淀粉组成的冷冻保护剂,通过-80℃低温冰箱转液氮阶梯降温法冷冻保存造血干细胞与传统10%二甲基亚砜作为冷冻保护剂用程序降温的方法取得一样效果,操作简便易于临床应用。

BACKGROUND： During the cryopreservation of hematopoietic stem cells,there are many influential factors,such as cooling rate,storage temperature,and cryoprotectant combination.There is a controversy in the cryopreservation methods.OBJECTIVE： To investigate the differences between -80 ℃ ladder-style freezing from low temperature refrigerator to liquid nitrogen method and programmed cooling method in preserving peripheral blood stem cells （PBSCs）.METHODS： Collected PBSCs were divided into two groups,and respectively cryopreserved by using -80 ℃ ladder-style freezing from low temperature refrigerator to liquid nitrogen method and programmed cooling method.RESULTS AND CONCLUSION： There was no significant difference between these two frozen samples of PBSCs after recovery in the aspects of trypan blue exclusion rate,recovery,apoptosis rate and mortality （P 0.05）.These findings indicate that the -80 ℃ ladder-style freezing from low temperature refrigerator to liquid nitrogen method using the cryoprotectant containing 5% dimethyl sulfoxide,4% albumin and 3% hydroxyethyl starch can yield the same effect as same as the programmed cooling method using 10% DMSO as cryoprotectant in the aspect of PBSCs cryopreservation,and the former one is more convenient and more suitable for clinical application.