大规模猪肝细胞低温保存方法的建立

ESTABLISHMENT OF METHOD OF LARGE-SCALE CRYOPRESERVATION OF PORCINE HEPATOCYTES

目的 建立大规模猪肝细胞的低温保存方法以满足生物型人工肝治疗的需要。方法 用酶法从中国实验用小型猪肝脏分离出猪肝细胞 ;加入本实验室配制的含有 10 %二甲基亚砜 (dimethylsulfoxide,DMSO)的营养液中 ;分别采用两种降温方法使 (12 )× 10 10 猪肝细胞保存在 - 196℃液氮中 ;1个月后复温 ,观察在同一条件下培养不同时间后猪肝细胞形态、存活率和白蛋白、尿素、葡萄糖合成功能及对利多卡因的转化功能。结果 用两种降温方法保存的肝细胞 ,复温后细胞的存活率均较高 (梯度降温组和程控降温组存活率分别较冻存前降低了 4 7%和 8 6 %) ,其中前者肝细胞的尿素、葡萄糖合成功能较后者高。结论 所建立的冻存方法简单易行 ,可保存大量的猪肝细胞。

Objective To establish a method of large-scale cryopreservation of porcine hepatocytes to meet the need of the biological artificial liver. Methods Porcine hepatocytes were isolated from Chinese miniature swines by collagenase disgestion. The cells were suspended in self-made nutrient solution supplemented with 10% DMSO. The hepatocytes (1×10 10~2×10 10) were stored in liquid nitrogen (-196℃) after being treated with two kinds of freezing methods. The cryopreserved hepatocytes were thawed in 39℃ water after one month and cultured. The morphology, viability and the function of thawed hepatocytes were observed. Results The cell viability of cryobreserved hepatocytes after both freezing methods were high. However, synthesis of urea nitrogen and glucose was higher in stepwise temperature lowering group compared with programmed temperature lowering group. Conclusion The results indicate that the cryopreservation method is feasible and simple, large amount porcine hepatocytes can be cryopreserved.