用于生物人工肝支持系统的猪肝细胞的分离与短期培养。

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system.

作者信息

Li Jun, Li Lan-Juan, Chao Hong-Cui, Yang Qian, Liu Xiao-Li, Sheng Ji-Fang, Yu Hai-Ying, Huang Jian-Rong

机构信息

Department of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2005 May;4(2):249-53.

PMID:15908324

Abstract

BACKGROUND

The demand for the clinical use of hepatocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes.

METHODS

The domestic extracorporeal circulating perfusion apparatus was used to isolate and harvest swine hepatocytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and observed by the trypan blue exclusion test, biochemical measurements, phase contrast microscopy and transmission electron micrography (TEM).

RESULTS

The total yield of isolated hepatocytes reached to 1.5(+/-0.4)X10(10) per liver with a viability of 92(+/-5)%, and the purity of hepatocytes reached to 98%. Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly.

CONCLUSION

This method of procuring swine hepatocytes could get high quality cells with active metabolic function.

摘要

背景

临床对肝细胞的需求日益增加。本研究的目的是开发一种获取高质量猪肝细胞的方法，并评估新鲜分离和培养的肝细胞的状态。

方法

使用国产体外循环灌注装置，采用乙二胺四乙酸（EDTA）和胶原酶两步灌注法分离和收获猪肝细胞。通过台盼蓝排斥试验、生化检测、相差显微镜和透射电子显微镜（TEM）对新鲜分离和培养的细胞的活力、功能和形态进行评估和观察。

结果

每只肝脏分离的肝细胞总产量达到1.5（±0.4）×10¹⁰，活力为92（±5）%，肝细胞纯度达到98%。分离后立即进行相差显微镜和TEM观察，发现未受损的肝细胞呈明亮、半透明的球形，边界特征明显。培养24小时后，培养液中丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）、白蛋白（ALB）、肌酐（Cr）和血尿素氮（BUN）的浓度显著下降。