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Safe and Efficient Gene Transfer into Porcine Hepatocytes Using Sendai Virus‐Cationic Liposomes for Bioartificial Liver Support
Author(s) -
Kawashita Yujo,
Ohtsuru Akira,
Fujioka Hikaru,
Kamohara Yukio,
Kawazoe Yasushi,
Sugiyama Nozomu,
Eguchi Susumu,
Kuroda Hiroaki,
Furui Junichiro,
Yamashita Shunichi,
Kaneda Yasufumi,
Kanematsu Takashi
Publication year - 2000
Publication title -
artificial organs
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.684
H-Index - 76
eISSN - 1525-1594
pISSN - 0160-564X
DOI - 10.1046/j.1525-1594.2000.06631.x
Subject(s) - sendai virus , bioartificial liver device , microcarrier , transfection , liposome , microbiology and biotechnology , hepatocyte , luciferase , chemistry , genetic enhancement , cationic liposome , cell , biology , biochemistry , gene , in vitro
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 × 10 5 cells and exposure duration of 90 min. Under these conditions, β‐gal and luciferase genes were transduced to 2.5 × 10 8 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.