Safe and Efficient Gene Transfer into Porcine Hepatocytes Using Sendai Virus-Cationic Liposomes for Bioartificial Liver Support

: Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by β-galactosidase (β-gal) staining, fluorescence activated cell sorting analysis for β-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 μg of deoxyribonucleic acid with 200 μg of lipid/2 × 105 cells and exposure duration of 90 min. Under these conditions, β-gal and luciferase genes were transduced to 2.5 × 108 isolated porcine hepatocytes following attachment to the beads. Positive β-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.