Genetic modification of hepatocytes towards hepatocyte transplantation and liver tissue engineering.
Cell-based therapies, including liver tissue engineering following hepatocyte transplantation, have therapeutic potential for several types of liver diseases. Modifications in the methodology to manipulate the donor hepatocytes in a more simple and timely manner prior to transplantation would enhance the therapeutic efficacy of this procedure. Conventional approach for vector-mediated gene transduction to the isolated hepatocytes has been performed under primary culture conditions that routinely require several days to complete. In our study, we have established a clinically feasible approach that requires only 1 h of infection time with an adenoviral vector system that results in an extremely efficient transduction efficiency (> 80%). To optimize transduction efficiency and sustain normal cellular function, we determined that the isolated hepatocytes should be maintained in UW solution as a suspension medium and infected with adenoviral vectors (Ad) for no more than 1 h at a MOI of 1. To establish if the isolated hepatocytes could be used as a source for cell-based therapies, we transplanted the Ad-transduced hepatocytes into the liver or under the kidney capsule. When the cells were transplanted into the liver, Ad-transduced hepatocytes cultured in suspension conditions were found to have a significantly higher survival rate (p < 0.01) than Ad-transduced hepatocytes cultured under standard conditions. We also confirmed that these Ad-transduced hepatocytes have ability to survive long term and were able to engineer a biologically active hepatic tissue under the kidney capsule. Finally, we obtained high level of transduction into canine, porcine, and human isolated hepatocytes in a suspension solution mixed with Ad. In conclusion, the present studies demonstrate that isolated hepatocytes could be genetically modified using Ad when kept in a suspension solution. For this reason, this cell-modified technique could be used for the treatment of liver-targeted diseases and/or disorders.
Kuge
Hiroyuki
H
Department of Surgery, Nara Medical University, Nara, Japan.
Ohashi
Kazuo
K
Yokoyama
Takashi
T
Kanehiro
Hiromichi
H
Hisanaga
Michiyoshi
M
Koyama
Fumikazu
F
Bumgardner
Ginny L
GL
Kosai
Ken-Ichiro
K
Nakajima
Yoshiyuki
Y
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D005822:Genetic Vectors / Q000235:genetics; D006085:Graft Survival; D022781:Hepatocytes / Q000502:physiology / Q000637:transplantation* / Q000821:virology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000166:cytology; D051379:Mice / Q000235:genetics; D008815:Mice, Inbred Strains / Q000235:genetics; D008822:Mice, Transgenic / Q000235:genetics; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods