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Cell Tissue Res (2004) 317: 4556
DOI 10.1007/s00441-004-0894-6REGULAR ARTICLEPeggy Mller . Hendryk Aurich . Ralph Wenkel .
Ines Schffner . Ilona Wolff . Jens Walldorf .
Wolfgang E. Fleig . Bruno ChristSerum-free cryopreservation of porcine hepatocytesReceived: 19 February 2004 / Accepted: 5 April 2004 / Published online: 5 June 2004
# Springer-Verlag 2004Abstract The use of porcine hepatocytes in xenotransplantation, bioartificial liver support or pharmacological
approaches demands serum-free cryopreservation protocols yielding high quality, viable, functional hepatocytes.
Here, primary porcine hepatocytes were frozen without
serum in liquid nitrogen by the use of a computer-assisted
freezing device. After thawing, more than 90% of the
initial hepatocytes were lost, in part because of damage to
genomic DNA. When cryoprotectants were used, the loss
was lowered to 70% of the initial cell number; 90% of the
remaining cells excluded trypan blue indicating a high
degree of viability. Cells were seeded serum-free onto
collagen-coated plastic dishes to determine proliferation
and retainment of specific functions representing prominent features of hepatocytes in vivo. Whereas no cells
adhered to the substratum effectively in conventional
culture medium, the addition of conditioned medium
derived from hepatic non-parenchymal cells improved
attachment. Cells proliferated, retained hepatocyte-specific
functions, such as urea production and cytochrome P450
activity, and expressed liver-specific genes to levels
observed in non-cryopreserved hepatocytes. Thus,
serum-free cryopreserved primary porcine hepatocytes
may serve as a valid source of cells for downstream
applications. The cells seem to function adequately when
an appropriate environment is chosen for recovery after
cryopreservation, an ultimate demand for the clinical
application of human hepatocytes.Keywords Comet assay . Cryopreservation .
Hepatocytes . Transplantation . Liver-specific functions .
Pig . Rat (male Wistar)P. Mller . H. Aurich . I. Schffner . I. Wolff . J. Walldorf .W. E. Fleig . B. ChristMolecular Hepatology Unit, First Department of Internal
Medicine, Center for Applied Medical and Human Biological
Research, Martin Luther University at Halle-Wittenberg,
Halle/Saale, GermanyP. Mller (*)
Molekulare Hepatologie, Klinik und Poliklinik fr Innere
Medizin I, Zentrum fr Angewandte Medizinische und
Humanbiologische Forschung,
Heinrich-Damerow-Strae 1,06097 Halle/Saale, Germanye-mail: [email protected].: +49-345-5522871Fax: +49-345-5522864R. WenkelInstitute of Animal Breeding and Husbandry with Veterinary
Clinic, Martin Luther University at Halle-Wittenberg,
Halle/Saale, GermanyIntroductionBecause of the limited availability of donor organs, the
transplantation of allogeneic hepatocytes is increasingly
becoming a feasible alternative to orthotopic...