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A Simple and Highly Effective Method for the Stable Transduction of Uncultured Porcine Hepatocytes Using Lentiviral Vector

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Abstract:

Gene therapy is an attractive approach for the treatment of a wide spectrum of liver diseases. Lentiviral vectors allow the stable integration of transgenes into the genome of nondividing differentiated cells including hepatocytes and could provide long-lasting expression of a therapeutic gene. To develop such approaches, preclinical studies in large animal models such as pigs are necessary to evaluate the feasibility and safety of stable lentiviral integration and long-term vector expression. In addition, effective lentivector-mediated gene transfer onto porcine hepatocytes could advance in cell-based therapies for acute liver failure. To investigate this issue, porcine hepatocytes were transduced in suspension immediately after their isolation in University of Wisconsin (UW) solution containing vitamin E. Up to 80% of hepatocytes stably expressed a GFP transgene after a single exposure to lentiviral vector coding for GFP under the control of either liver-specific or ubiquitous promoters. Moreover, porcine hepatocytes cryopreserved in UW solution containing fetal bovine serum, dimethyl sulfoxide, and vitamin E remained highly transducible with lentiviral vector after thawing. When thawed, transduced in suspension, and immediately transplanted into the spleen of immunodeficient mice, ex vivo lentivirally transgene marked xenogeneic hepatocytes were detected in murine liver. We demonstrated that porcine hepatocytes are highly susceptible to lentiviral vector and describe an easy methodology to efficiently, rapidly, and stably introduce transgenes into uncultured porcine hepatocytes.

Keywords: Cryopreservation; Gene transfer; Lentiviral vector; Liver; Porcine hepatocytes; Transplantation

Document Type: Research Article

DOI: https://doi.org/10.3727/000000005783982828

Affiliations: 1: Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, CH 1211, Switzerland 2: Cell Genesys Inc., South San Francisco, CA 94080, USA 3: Department of Visceral Surgery, Faculty of Medicine, University of Geneva, Geneva, CH1211, Switzerland 4: Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, CH 1211, Switzerland, Ecole Polytechnique Fédérale de Lausanne, Lausanne, CH 1015, Switzerland

Publication date: 2005-07-01

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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