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 beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-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 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-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.
Kawashita
Y
Y
Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, Japan.
Ohtsuru
A
A
Fujioka
H
H
Kamohara
Y
Y
Kawazoe
Y
Y
Sugiyama
N
N
Eguchi
S
S
Kuroda
H
H
Furui
J
J
Yamashita
S
S
Kaneda
Y
Y
Kanematsu
T
T
D000818:Animals; D002478:Cells, Cultured; D003094:Collagen; D005260:Female; D018014:Gene Transfer Techniques; D022781:Hepatocytes / Q000378:metabolism; D008081:Liposomes; D019164:Liver, Artificial; D008863:Microspheres; D018108:Respirovirus / Q000235:genetics; D013552:Swine; D014161:Transduction, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D008081:Liposomes; D003094:Collagen; D001616:beta-Galactosidase