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Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies

Authors

  • Stefan A. Hoffmann,

    1. Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; telephone: +49-30-450-552-501; fax: +49-30-450-559-909
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  • Ursula Müller-Vieira,

    1. Pharmacelsus GmbH, Science Park 2, Saarbrücken, Germany
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  • Klaus Biemel,

    1. Pharmacelsus GmbH, Science Park 2, Saarbrücken, Germany
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  • Daniel Knobeloch,

    1. Cytonet GmbH & Co. KG, Niederlassung Heidelberg, Heidelberg, Germany
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  • Sandra Heydel,

    1. ElexoPharm GmbH, Im Stadtwald, Saarbrücken, Germany
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  • Marc Lübberstedt,

    1. Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; telephone: +49-30-450-552-501; fax: +49-30-450-559-909
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  • Andreas K. Nüssler,

    1. BG Unfallklinik Tübingen, Eberhard Karls Universität Tübingen, Tübingen, Germany
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  • Tommy B. Andersson,

    1. DMPK Innovative Medicines, AstraZeneca R&D, Mölndal, Sweden
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  • Jörg C. Gerlach,

    1. McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Philadelphia
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  • Katrin Zeilinger

    Corresponding author
    1. Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; telephone: +49-30-450-552-501; fax: +49-30-450-559-909
    • Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; telephone: +49-30-450-552-501; fax: +49-30-450-559-909.
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Abstract

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/106 cells followed by a period of stable conversion of about 100 pmol/h/106 cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver. Biotechnol. Bioeng. 2012; 109: 3172–3181. © 2012 Wiley Periodicals, Inc.

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