生物人工肝乳猪肝细胞的聚集培养、冻存及形态、功能观察  

Morphology, viability and function of suckling pig hepatocytes in spheroid culture and cryopreservation for bioartificial liver system

作　　者：秦爱兰[1] 周霞秋[1] 俞红[1] 谢青[1] 张韡[1] 郭清[1] 

机构地区：[1]上海第二医科大学附属瑞金医院传染病科,200025

出　　处：《肝脏》2003年第3期9-11,72,共4页Chinese Hepatology

基　　金：上海市自然科学基金资助 ;编号 :0 0 4 1 1 90 4 4

摘　　要：目的　为构建生物人工肝进行肝细胞的准备。方法　采用胶原酶半原位灌流法分离单个乳猪肝细胞 ,并对其活力、单层、聚集培养及冻存复苏后培养的白蛋白、尿素合成功能进行检测。结果　采用本方法从每头乳猪中分离到的单个肝细胞数为 ( 3 .1± 1.5 )× 10 10 ,活性超过 95 %。在加入激素和生长因子的培养基中单层培养时 ,肝细胞功能良好 ,可维持 2周左右 ,而在未加入激素和生长因子的培养基中肝细胞虽能存活 1周 ,但功能于 2 4h后即丧失。球形聚集培养可实现肝细胞的大量培养 ,且生物学活性较单层培养显著提高。冻存复苏后肝细胞活力仍超过 90 % ,肝细胞功能与新鲜分离培养的肝细胞功能间无明显差异。结论　采用胶原酶半原位灌注法所得单个乳猪肝细胞基本满足构建生物人工肝对肝细胞数量的要求。聚集培养接种密度大 ,细胞生物学活性高 ,可用于构建生物人工肝。冻存复苏后肝细胞仍功能良好。Objective Hepatic failure is a serious clinical syndrome. A possible solution to this problem is by bioartificial liver system, of which its preparation, isolation and culture of pig hepatocytes in large scale are needed. This study was aimed to evaluate the changes in morphology, viability and function of pig hepatocytes during spheroid culture and cryopreservation.Methods Hepatocyte isolation was performed by modified two-step half in situ collagenase perfusion via portal vein. The morphology, viability, protein and urea synthesis were compared among the monolayer culture, speroid culture and cryopreservation.Results The total number of hepatocytes isolated from a pig was 3.1±1.5(×10 10) and the viability was more than 95%。 The function of the cultured hepatocytes in the medium supplemented with dexamethasone, insulin, HGF, EGF, FBS was stabilized for about two weeks. The spheroid culture was superior to monolayer culture in the functional aspect of hepatocytes. After cryopreservation, the viability of hepatocytes was still more than 90%, and the function was the same as that prior to cryopreservation.Conclusion The viability and yield of hepatocytes are sufficient for clinical use of bioartificial liver system. The function of the hepatocytes seems to be better in the spheroid culture than that in the monolayer culture. The hepatocytes after cryopreservation are still suitable for the usage of bioartificial liver system.

关 键 词：生物人工肝 乳猪 肝细胞 细胞聚集 细胞培养 冷冻保存 

分 类 号：R318.14[医药卫生—生物医学工程]