Abstract
Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the “gold standard” for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h. The use of animal- and human-derived hepatocytes allows interspecies comparisons of metabolic properties. In contrast with microsomes, which are easily prepared from human liver tissue and can be stored in liquid nitrogen with minimal loss of functionality, cryopreservation of isolated human hepatocytes has been shown to be more difficult: after thawing losses of cell viability and biotransformation capacity occur. We provide general recommendations for the appropriate use of hepatocytes in suspension for pharmaco-toxicological studies. We also provide protocols for the cryopreservation of freshly isolated hepatocytes and their handling on thawing.
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Li, A. P., Gorycki, P. D., Hengstler, J. G., et al. (1999) Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel. Chem. Biol. Interact. 121, 117–123.
Bachmann, K. A. and Ghosh, R. (2001) The use of in vitro methods to predict in vivo pharmacokinetics and drug interactions. Curr. Drug Metab. 2, 299–314.
Elaut, G., Papeleu, P., Rogiers, V., and Vanhaecke, T. (2002) Practical aspects of in vitro biotransformation studies during early drug development, in Recent Research Developments in Drug Metabolism and Disposition, vol. 1 (Pandalai, S. G., ed.), Transworld Research Network, Kerala, India, pp. 167–198.
Naritomi, Y., Terashita, S., Kagayama, A., and Sugiyama, Y. (2003) Utility of hepatocytes in predicting drug metabolism: comparison of hepatic intrinsic clearance in rats and humans in vivo and in vitro. Drug Metab. Dispos. 31, 580–588.
Hengstler, J. G., Utesch, D., Steinberg, P., et al. (2000) Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab. Rev. 32, 81–118.
Li, A. P. (1997) Primary hepatocyte cultures as an in vitro experimental model for the evaluation of pharmacokinetic drug-drug interactions. Adv. Pharmacol. 43, 103–127.
Elaut, G., Török, G., Vinken, M., et al. (2002) Major phase I biotransformation pathways of Trichostatin A in rat hepatocytes and in rat and human liver microsomes. Drug Metab. Dispos. 30, 1320–1328.
Houston, J. B. (1994) Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 47, 1469–1479.
Ito, K., Iwatsubo, T., Kanamitsu, S., Nakajima, Y., and Sugiyama, Y. (1998) Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport. Annu. Rev. Pharmacol. Toxicol. 38, 461–499.
Skett, P. (1994) Problems in using isolated and cultured hepatocytes or xenobiotic metabolism/metabolism-based toxicity testing—solutions? Toxicol. In Vitro 8, 491–504.
Carlile, D. J., Stevens, A. J., Ashforth, E. I. L., Waghela, D., and Houston, J. B. (1998) In vivo clearance of ethoxycoumarin and its prediction from in vitro systems: use of drug depletion and metabolite formation methods in hepatic microsomes and isolated hepatocytes. Drug Metab. Dispos. 26, 216–221.
Guillouzo, A., Rialland, L., Fautrel, A., and Guyomard, C. (1999) Survival and function of isolated hepatocytes after cryopreservation. Chem. Biol. Interact. 122, 7–16.
Alexandre, E., Viollon-Abadie, C., David, P., et al. (2002) Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiology 44, 103–113.
Lau, Y. Y., Sapidou, E., Cui, X., White, R. E., and Cheng, K. C. (2002) Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. Drug Metab. Dispos. 30, 1446–1454.
Houle, R., Raoul, J., Lévesque, J. F., Pang, K. S., Nicoll-Griffith, D. A., and Silva, J. M. (2003) Retention of transporter activities in cryopreserved, isolated rat hepatocytes. Drug Metab. Dispos. 31, 447–451.
Chesné, C., Guyomard, C., Fautrel, A., et al. (1993) Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation. Hepatology 18, 406–414.
Seglen, P. O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83.
Wiggs, A. J., Philips, J. W., and Berry, M. N. (2003) Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°C. Liver Int. 23, 201–211.
Cross, D. M. and Bayliss, M. K. (2000) A commentary on the use of hepatocytes in drug metabolism studies during drug discovery and development. Drug Metab. Rev. 32, 219–240.
Gebhardt, R., Hengstler, J. G., Müller, D., et al. (2003) New hepatocyte in vitro systems for drug metabolism: application in basic research and drug development, standard operation procedures. Drug Metab. Rev. 35, 145–213.
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467–477.
Elaut, G., Vanhaecke, T., Heyden, Y. V., and Rogiers, V. (2005) Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium. Biochem. Pharmacol. 16, 1829–1838.
Blaauboer, B. J., Boobis, A. R., Castell, J. V., et al. (1994) The practical applicability of hepatocyte cultures in routine testing. Altern. Lab. Animal. 22, 231–241.
Gulden, M., Morchel, S., and Seibert, H. (2001) Factors influencing nominal effective concentrations of chemical compounds in vitro: cell concentration. Toxicol. In Vitro 15, 233–243.
McGinnity, D. F. and Riley, R. J. (2001) Predicting drug pharmacokinetics in humans from in vitro metabolism studies. Biochem. Soc. Trans. 29, 135–139.
Chauret, N., Gauthier, A., and Nicoll-Griffith, D. A. (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metab. Dispos. 26, 1–4.
Easterbrook, J., Lu, C., Sakai, Y., Li, A. P. (2001) Effects of organic solvents on the activities of cytochrome P450 isoforms, UDP-dependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes. Drug Metab. Dispos. 29, 121–144.
Ulgen, M. (1999) Chemical artifacts in drug metabolism research. Boll. Chim. Farm. 138, 388–396.
de Sousa, G., Langouët, S., Nicolas, F., et al. (1991) Freshly isolated or cryopreserved human hepatocytes in primary culture: influence of drug metabolism on hepatotoxicity. Toxicol. In Vitro 5, 483–486.
Dou, M., de Sousa, G., Lacarelle, B., et al. (1992) Thawed human hepatocytes in primary culture. Cryobiology 29, 454–469.
Diener, B., Traiser, M., Arand, M., et al. (1994) Xenobiotic metabolizing enzyme activities in isolated and cryopreserved human liver parenchymal cells. Toxicol. In Vitro 8, 1161–1166.
Adams, R. M., Wang, M., Crane, A. M., Brown, B., Darlington, G. J., and Ledley, F. D. (1995) Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential. Cell Transplant. 4, 579–586.
Darr, T. B., and Hubel, A. (2001) Postthaw viability of precultured hepatocytes. Cryobiology 41, 11–20.
Swales, N. J. and Utesch, D. (1998) Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions. Xenobiotica 28, 937–948.
Ratanasavanh, D., Beaune, P., Morel, F., Flinois, J. P., Guengerich, F. P., and Guillouzo, A. (1991) Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age. Hepatology 13, 1142–1151.
Acknowledgments
This work was supported by grants from the Fund of Scientific Research Flanders (FWO), Belgium; the Research Council (OZR) of the Vrije Universiteit Brussel, Belgium, and the EU Sixth Framework Program.
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Elaut, G. et al. (2006). Hepatocytes in Suspension. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:255
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DOI: https://doi.org/10.1385/1-59259-998-2:255
Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-59259-998-1
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