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Serum-free cryopreservation of porcine hepatocytes

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Abstract

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.

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Acknowledgements

The excellent technical assistance of Sabine Ebensing and Madlen Hempel is greatly appreciated.

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Authors and Affiliations

  1. Molecular Hepatology Unit, First Department of Internal Medicine, Center for Applied Medical and Human Biological Research, Martin Luther University at Halle-Wittenberg, Halle/Saale, Germany

    Peggy Müller, Hendryk Aurich, Ines Schäffner, Ilona Wolff, Jens Walldorf, Wolfgang E. Fleig & Bruno Christ

  2. Institute of Animal Breeding and Husbandry with Veterinary Clinic, Martin Luther University at Halle-Wittenberg, Halle/Saale, Germany

    Ralph Wenkel

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  1. Peggy Müller
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  2. Hendryk Aurich
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  3. Ralph Wenkel
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  4. Ines Schäffner
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Correspondence to Peggy Müller.

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Müller, P., Aurich, H., Wenkel, R. et al. Serum-free cryopreservation of porcine hepatocytes. Cell Tissue Res 317, 45–56 (2004). https://doi.org/10.1007/s00441-004-0894-6

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