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


CONCLUSIONS
This method of procuring swine hepatocytes could get high quality cells with active metabolic function.
Li
Jun
J
Department of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. lilj@zjwst.gov.cn
Li
Lan-Juan
LJ
Chao
Hong-Cui
HC
Yang
Qian
Q
Liu
Xiao-Li
XL
Sheng
Ji-Fang
JF
Yu
Hai-Ying
HY
Huang
Jian-Rong
JR
D000818:Animals; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D017690:Cell Transplantation / Q000379:methods*; D005260:Female; D022781:Hepatocytes / Q000166:cytology*; D019164:Liver, Artificial; D008297:Male; D023421:Models, Animal; D012680:Sensitivity and Specificity; D013552:Swine