Abstract
The liver performs a multitude of functions including the regulation of carbohydrate, fat, and protein metabolism, the detoxification of endo- and xenobiotics, and the synthesis and secretion of plasma proteins and bile. Isolated hepatocytes constitute a useful technique for studying liver function in an in vitro setting. Here we describe a method for the isolation of hepatocytes from adult mouse liver. The principle of the method is the two-step collagenase perfusion technique which involves sequential perfusion of the liver with ethylenediaminetetraacetic acid and collagenase. Following isolation, the cells can either be used for short-term studies or, alternatively, maintained in culture for prolonged periods to study long-term changes in gene expression. The protocol for mouse hepatocyte isolation may be applied to both normal and transgenic mice.
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Berry, M. N., Halls, H. J., and Grivell, M. B. (1995) Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions. Life Sci. 51, 1–16.
Parrish, A. R., Gandolfi, A. J., and Brendel, K. (1995) Precision-cut tissue slices: applications in pharmacology and toxicology. Life Sci. 57, 1887–1901.
Mitaka, T. (1998) The current status of primary hepatocyte culture. Int. J. Exp. Pathol. 79, 393–409.
Berry, M. N., and Friend, D. S. (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43, 506–520.
Schreiber, G., and Schreiber, M. (1973) The preparation of single cell suspensions from liver and their use for the study of protein synthesis. Subcell. Biochem. 2, 307–353.
Howard, R. B., Christensen, A. K., Gibbs, F. A., and Pesch, L. A. (1967) The enzymatic preparation of isolated intact parenchymal cells from rat liver. J. Cell Biol. 35, 675–684.
Seglen, P. O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83.
Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Chacko, J., and Trump, B. F. (1981) Mouse liver cell culture. I. Hepatocyte isolation. In Vitro 17, 913–925.
Troyanovsky, S. (2005) Cadherin dimers in cell–cell adhesion. Eur. J. Cell Biol. 84, 225–233.
Takahashi, S., and Seifter, S. (1970) Dye-sensitized photo-inactivation of collagenase A. Biochim. Biophys Acta 214, 556–558.
Berry, M. N., Grivell, A. R., Grivell, M. B., and Phillips, J. W. (1997) Isolated hepatocytes – past, present and future. Cell Biol. Toxicol. 13, 223–233.
Howard, R. B., Lee, J. C., and Pesch, L. A. (1973) The fine structure, potassium content, and respiratory activity of isolated rat liver parenchymal cells prepared by improved enzymatic techniques. J. Cell Biol. 57, 642–658.
Olinga, P., Merema, M., Hof, I. H., de Jong, K. P., Slooff, M. J., Meijer, D. K., and Groothuis, G. M. (1998) Effect of human liver source on the functionality of isolated hepatocytes and liver slices. Drug Metab. Dispos. 26, 5–11.
Denizot, F., and Lang, R. (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271–277.
Behrsing, H. P., Vickers, A. E., and Tyson, C. A. (2003) Extended rat liver slice survival and stability monitored using clinical biomarkers. Biochem. Biophys. Res. Commun. 2003(312), 209–213.
Agius, L. (1987) Human liver in vitro techniques for metabolic studies. Baillieres Clin. Endocrinol. Metab. 1, 999–1021.
Michalopoulos, G. K. (1990) Liver regeneration: molecular mechanisms of growth control. FASEB J. 4, 176–187.
Wang, Y. J., Liu, H. L., Guo, H. T., Wen, H. W., and Liu, J. (2004) Primary hepatocyte culture in collagen gel mixture and collagen sandwich. World J. Gastroenterol. 10, 699–702.
Bhatia, S. N., Balis, U. J., Yarmush, M. L., and Toner, M. (1998) Microfabrication of hepatocyte/fibroblast co-cultures: role of homotypic cell interactions. Biotechnol. Prog. 14, 378–387.
Kang, Y. H., Berthiaume, F., Nath, B. D., and Yarmush, M. L. (2004) Growth factors and nonparenchymal cell conditioned media induce mitogenic responses in stable long-term adult rat hepatocyte cultures. Exp. Cell Res. 293, 239–247.
Sidelmann, U. G., Cornett, C., Tjornelund, J., and Hansen, S. H. (1996) A comparative study of precision cut liver slices, hepatocytes, and liver microsomes from the Wistar rat using metronidazole as a model substance. Xenobiotica 26, 709–722.
Cervenkova, K., Belejova, M., Vesely, J., Chmela, Z., Rypka, M., Ulrichova, J., Modriansky, M. et al. (2001) Cell suspensions, cell cultures, and tissue slices – important metabolic in vitro systems. Biomed. Pap. Med. Fac. Univ. Palacky. Olomouc. Czech. Repub. 145, 57–60.
Kojima, T., Mitaka, T., Shibata, Y., and Mochizuki, Y. (1995) Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes. J. Cell Sci. 108, 2771–2780.
Miyazaki, M., Mars, W. M., Runge, D., Kim, T. H., Bowen, W. C., and Michalopoulos, G. K. (1998) Phenobarbital suppresses growth and accelerates restoration of differentiation markers of primary culture rat hepatocytes in the chemically defined hepatocyte growth medium containing hepatocyte growth factor and epidermal growth factor. Exp. Cell Res. 241, 445–457.
Lilja, H., Kamohara, Y., Neuman, T., Demetriou, A. A., and Rozga, J. (1999) Transforming growth factor beta1 helps maintain differentiated functions in mitogen-treated primary rat hepatocyte cultures. Mol. Cell Biol. Res. Commun. 1, 188–195.
Runge, D., Runge, D. M., Jager, D., Lubecki, K. A., Beer Stolz, D., Karathanasis, S., Kietzmann, T. et al. (2000) Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem. Biophys. Res. Commun. 269, 46–53.
Mizuguchi, T., Hui, T., Palm, K., Sugiyama, N., Mitaka, T., Demetriou, A. A., and Rozga, J. (2001) Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells. J. Cell. Physiol. 189, 106–119.
Katsura, N., Ikai, I., Mitaka, T., Shiotani, T., Yamanokuchi, S., Sugimoto, S., Kanazawa, A. et al. (2002) Long-term culture of primary human hepatocytes with preservation of proliferative capacity and differentiated functions. J. Surg. Res. 106, 115–123.
Iocca, H. A., and Isom, H. C. (2003) Tumor necrosis factor-alpha acts as a complete mitogen for primary rat hepatocytes. Am. J. Pathol. 163, 465–476.
Vanhaecke, T., Henkens, T., Kass, G. E., and Rogiers, V. (2004) Effect of the histone deacetylase inhibitor trichostatin A on spontaneous apoptosis in various types of adult rat hepatocyte cultures. Biochem. Pharmacol. 68, 753–760.
Gardmo, C., Kotokorpi, P., Helander, H., and Mode, A. (2005) Transfection of adult primary rat hepatocytes in culture. Biochem. Pharmacol. 69, 1805–1813.
Siendones, E., Jimenez-Gomez, Y., Montero, J. L., Gomez-Diaz, C., Villalba, J. M., and Muntane, J. (2005) PGE1 abolishes the mitochondrial-independent cell death pathway induced by D-galactosamine in primary culture of rat hepatocytes. J. Gastroenterol. Hepatol. 20, 108–116.
Noreault, T. L., Jacobs, J. M., Nichols, R. C., Trask, H. W., Wrighton, S. A., Sinclair, P. R., and Sinclair, J. F. (2005) Mechanism of arsenite-mediated decreases in CYP3A23 in rat hepatocytes. Biochem. Biophys. Res. Commun. 333, 1211–1217.
Kimura, M., and Ogihara, M. (2005) Effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur. J. Pharmacol. 510, 167–180.
Abdelmegeed, M. A., Carruthers, N. J., Woodcroft, K. J., Kim, S. K., and Novak, R. F. (2005) Acetoacetate Induces CYP2E1 Protein and Suppresses CYP2E1 mRNA in Primary Cultured Rat Hepatocytes. J. Pharmacol. Exp. Ther. 315, 203–213.
Schmidt, M., Schmitz, H. J., Baumgart, A., Guedon, D., Netsch, M. I., Kreuter, M. H., Schmidlin, C. B. et al. (2005) Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture. Food Chem. Toxicol. 43, 307–314.
Li, W.-C., Ralphs, K. L., Slack, J. M. W., and Tosh, D. (2007) Keratinocyte serum-free medium maintains long term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors. Int. J. Biochem. Cell Biol. 39, 541–554.
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Li, WC., Ralphs, K.L., Tosh, D. (2010). Isolation and Culture of Adult Mouse Hepatocytes. In: Ward, A., Tosh, D. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 633. Humana Press. https://doi.org/10.1007/978-1-59745-019-5_13
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DOI: https://doi.org/10.1007/978-1-59745-019-5_13
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