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Human Hepatocytes: Isolation, Culture, and Quality Procedures

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Human Cell Culture Protocols

Abstract

The use of isolated human liver cells in research and development has gained increasing interest during the past years. The possible application may vary between elucidation of new biochemical pathways in liver diseases, drug development, safety issues, and new therapeutic strategies up to direct clinical translation for liver support. However, the isolation of human liver cells requires a well-developed logistic network among surgeons, biologists, and technicians to obtain a high quality of cells. Our laboratories have been involved in various applications of human liver cells and we have long-lasting experiences in human liver cell isolation and their application in R&D. We here summarize the present protocol of our laboratories for cell isolation from normal resected liver tissue, the most common tissue available. In addition, we discuss the necessary network in the clinic and quality controls to maintain human liver cells in culture and the effect of 3D extracellular matrix in cultured cells which results in preservation of hepatocyte epithelial polarity in the form of bile canaliculi and repression of epithelial to mesenchymal transitions occurring in 2D cultures.

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References

  1. Branster, M. V., and Morton, R. K. (1957) Isolation of intact liver cells. Nature. 180, 1283–1284.

    Google Scholar 

  2. de Boer, J., and van Bavel, B. (2009) European “REACH” (Registration, Evaluation, Authorisation and Restriction of Chemicals) program. J Chromatogr A. 1216, 301.

    Google Scholar 

  3. Alexandre, E., Cahn, M., Abadie-Viollon, C., Meyer, N., Heyd, B., Mantion, G., et al. (2002) Influence of pre-, intra- and post-operative parameters of donor liver on the outcome of isolated human hepatocytes. Cell Tissue Bank. 3, 223233.

    Google Scholar 

  4. Lloyd, T. D., Orr, S., Patel, R., Crees, G., Chavda, S., Vadyar, H., et al. (2004) Effect of patient, operative and isolation factors on subsequent yield and viability of human hepatocytes for research use. Cell Tissue Bank. 5, 8187.

    Google Scholar 

  5. Seglen, P. O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol. 13, 2983.

    Google Scholar 

  6. Dorko, K., Freeswick, P. D., Bartoli, F., Cicalese, L., Bardsley, B. A., Tzakis, A., et al. (1994) A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation. Cell Transplant. 3, 387395.

    Google Scholar 

  7. Sharma, A. D., Cantz, T., Richter, R., Eckert, K., Henschler, R., Wilkens, L., et al. (2005) Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver. Am J Pathol. 167, 555564.

    Google Scholar 

  8. Beerheide, W., von Mach, M. A., Ringel, M., Fleckenstein, C., Schumann, S., Renzing, N., et al. (2002) Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells? Biochem Biophys Res Commun. 294, 1052–1063.

    Google Scholar 

  9. Hengstler, J. G., Brulport, M., Schormann, W., Bauer, A., Hermes, M., Nussler, A. K., et al. (2005) Generation of human hepatocytes by stem cell technology: definition of the hepatocyte. Expert Opin Drug Metab Toxicol. 1, 6174.

    Google Scholar 

  10. Williams, R. T. (1959) Detoxification Mecha­nisms. New York: John Wiley & Sons.

    Google Scholar 

  11. Parkinson, A. (1996) Biotransformation of xenobiotics. In Casarett and Doull’s Toxicology: the basic Science of Poinsons, ed. Klaassen, C. D. New York: McGraw-Hill. 113–186.

    Google Scholar 

  12. Nelson, D. R., Zeldin, D. C., Hoffman, S. M., Maltais, L. J., Wain, H. M., and Nebert, D. W. (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics. 14, 118.

    Google Scholar 

  13. Guengerich, F. P., and MacDonald, T. L. (1990) Mechanisms of cytochrome P-450 catalysis. FASEB J. 4, 24532459.

    Google Scholar 

  14. Pelkonen, O., Maenpaa, J., Taavitsainen, P., Rautio, A., and Raunio, H. (1998) Inhibition and induction of human cytochrome P450 (CYP) enzymes. Xenobiotica. 28, 12031253.

    Google Scholar 

  15. Donato, M. T., Jimenez, N., Castell, J. V., and Gomez-Lechon, M. J. (2004) Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos. 32, 699706.

    Google Scholar 

  16. Lin, J. H., and Lu, A. Y. (1997) Role of pharmacokinetics and metabolism in drug discovery and development. Pharmacol Rev. 49, 403449.

    Google Scholar 

  17. Hewitt, N. J., Lechon, M. J., Houston, J. B., Hallifax, D., Brown, H. S., Maurel, P., et al. (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev. 39, 159234.

    Google Scholar 

  18. Guillouzo, A., Morel, F., Fardel, O., and Meunier, B. (1993) Use of human hepatocyte cultures for drug metabolism studies. Toxicology. 82, 209219.

    Google Scholar 

  19. Rajan, N., Habermehl, J., Cote, M. F., Doillon, C. J., and Mantovani, D. (2006) Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc. 1, 27532758.

    Google Scholar 

  20. Decaens, C., Durand, M., Grosse, B., and Cassio, D. (2008) Which in vitro models could be best used to study hepatocyte polarity? Biol Cell. 100, 387398.

    Google Scholar 

  21. Rennert, B., and Melzig, M. F. (2002) Free fatty acids inhibit the activity of Clostridium histolyticum collagenase and human neutrophil elastase. Planta Med. 68, 767769.

    Google Scholar 

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Acknowledgments

This work was supported by the German Ministry of Research FKZ-BMBF 0315741 (Virtual Liver) and FZK-BMBF 01 GG 0732 (3-D TOX).

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Correspondence to Andreas K. Nussler .

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Knobeloch, D. et al. (2012). Human Hepatocytes: Isolation, Culture, and Quality Procedures. In: Mitry, R., Hughes, R. (eds) Human Cell Culture Protocols. Methods in Molecular Biology, vol 806. Humana Press. https://doi.org/10.1007/978-1-61779-367-7_8

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  • DOI: https://doi.org/10.1007/978-1-61779-367-7_8

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-366-0

  • Online ISBN: 978-1-61779-367-7

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