Woodchuck hepatocytes remain permissive for hepadnavirus infection and mouse liver repopulation after cryopreservation

Authors

  • Maura Dandri,

    1. Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany; the Departments of
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  • Martin R. Burda,

    1. Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany; the Departments of
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  • Andreas Gocht,

    1. Pathology,University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
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  • Eva Török,

    1. Hepatobiliary Surgery and Transplantation, and University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
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  • Dr. Jörg M. Pollok M.D.,

    Corresponding author
    1. Hepatobiliary Surgery and Transplantation, and University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
    • Medizinische Kernklinik und Poliklinik, Universitätsklinikum Hamburg Eppendorf, Martinistr. 52, D-20246, Hamburg, Germany. fax: (49) 40-42803-8065
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  • Charles E. Rogler,

    1. Medicine, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
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  • Hans Will,

    1. Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany; the Departments of
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  • Jörg Petersen

    Corresponding author
    1. Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany; the Departments of
    2. Albert Einstein College of Medicine, Marion Bessin Liver Research Center, Bronx, NY.
    • Medizinische Kernklinik und Poliklinik, Universitätsklinikum Hamburg Eppendorf, Martinistr. 52, D-20246, Hamburg, Germany. fax: (49) 40-42803-8065
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Abstract

Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to 60% of thawed cells attached to the plates. The appearance of covalently closed circular (ccc)DNA and of WHV-replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection, thus proving maintenance of a very high hepatocyte-specific differentiation status. Furthermore, transplantation of woodchuck hepatocytes into the liver of urokinase-type plasminogen activator (uPA)/recombination activation gene-2 (RAG-2) mice, a model of liver regeneration, demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.

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