Abstract
Primary culture of hepatocytes is an in vitro model widely used to investigate various aspects of liver physiology and pathology (1). In particular, such cultures have been extensively used for assessing the expression and function of drug-metabolizing enzymes including cytochromes P450 (CUP), drug metabolism, drug-drug interactions, and the mechanisms of cytotoxtcity and genotoxicity. Most of these studies have been carried out with rodent hepatocytes. However, because of species-specificity in both the regulation and activity of drug-metabolizing enzymes, extrapolation from animals to humans is not generally possible. For this reason, several groups have developed human hepatocyte culture systems (2–8). The technique used to isolate human hepatocytes is based on the two-step collagenase perfusion first mtroduced by Berry and Friend (9) and modified by Seglen (10). Originally performed in situ for obtaining hepatocytes from the adult rat, this technique of perfusion has been adapted to the ex vivo treatment of human liver tissue. The aim of this chapter is to describe the authors′ experience in the isolation of hepatocytes from human liver tissue and the preparation of short- and long-term cultures
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References
Gebhardt, R (1996) Special issue on international congress on hepatocytes applications in cell biology, toxicology and medicine. Cell Biol Toxicol. pp. 1–65
Maurel, P. (1996) The use of adult human hepatocytes in primary culture and other in vitro systems to investigate drug metabolism in man Adv Drug Del Rev 22, 105–132
Daujat, M., Pichard, L., Pineau, T., Fabre, G., Bonfils, C, and Maurel, P (1991) Induction protocols for cytochromes P450IIIA in vivo and in primary cultures of animal and human hepatocytes Methods Enzymol. 206, 345–353
Morel, F, Beaune, P, Ratanasavanh, D, Flinois, J P, Yang, C S, Guengench, F P., and Guillouzo, A (1990) Expression of cytochrome P-450 enzymes in cul tured human hepatocytes. Eur J Biochem 91, 437–444.
Gomez-Lechon, M. J., Castell, J, Guillen, I., O′Connor, E., Nakamura, T., Fabra, R, and Trullenque, R. (1995) Effects of hepatocyte growth factor on the growth and metabolism of human hepatocytes in primary culture. Hepatology 21, 1248–1254.
Ismail, T, Howl, J, Wheatley, M., McMaster, P, Neuberger, J M, and Strain, A J (1992) Growth of normal human hepatocytes n primary cultures Effect of hor-mones and growth factors on DNA synthesis. Hepatology 14, 1076–1082
Kostrubsky, V E, Strom, S. C, Wood, S G, Wrighton, S A, Sinclair, P R, and Sinclair, J. F (1995) Ethanol and isopentanol increase CYP3A and CYP2E in primary cultures of human hepatocytes Arch Biochem Bzophys 322, 516–520
Li, A. P., Rasmussen, A, Xu, L, and Kaminski, D. L. (1995) Rifampicin induc-tion of lidocaine metabolism in cultured human hepatocytes J Pharmacol Exp Ther 274, 673–677
Berry, M. N and Friend, D S (1969) High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 43, 506–520
Seglen, P. O (1976) Preparation of isolated rat liver cells Methods Cell Biol 18, 29–83
Isom, H. C. and Georgoff, I (1984) Quantitative assay for albumin-producing liver cells after Simian virus 40 transformation of rat hepatocytes maintained in chemically defined medium. Proc. Nat1 Acad. Sci USA. 81, 6378–6382
Lanford, R. E., Carey, K. D, Estlack, L. E., Smith, G C, and Hay, R. V. (1989) Analysis of plasma protein and lipoprotein synthesis in long-term primary cul-tures of baboon hepatocytes maintained in serum-free medium. In Vitro Cell Dev Biol 25, 174–182
Greuet, J, Pichard, L., Ourlin, J. C., Bonfils, C., Domergue, J, Le Treut, P., and Maurel, P. (1997) Effect of cell density and epidermal growth factor on the induc-ible expression of CY3A and CYP1A genes in human hepatocytes in primary culture Hepatology 25, 1166–1175
Daujat, M., Fabre, I., Diaz, D, Pichard, L., Fabre, G., Fabre, J M, Saint Aubert, B., and Maurel, P. (1990) Inducibility and expression of class IA and IIIA cyto-chromes P450 in primary cultures of adult human hepatocytes. Biochem Pharmacol (Life Sci Adv.) 9, 315–326.
Diaz, D, Fabre, I., Daujat, M., Saint Aubert, B., Bories, P, Michel, H, and Maurel, P (1990) Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450. Gastroenterology 99, 737–747
Dalet-Beluche, I, Boulenc, X., Fabre, G, Maurel, P, and Bonfils, C. (1992) Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. cross-reac-tivity with human forms. Eur. J. Biochem. 204, 641–648.
Pichard, L., Fabre, I., Daujat, M., Domergue, J, Joyeux, H., and Maurel, P. (1992) Effect of corticosteroids on the expression of cytochromes P450 and on cyclo-sporin A oxidase activity in primary cultures of human hepatocytes A401 Pharmacol. 41, 1047–1055.
Curi-Pedrosa, R., Daujat, M., Pichard, L., Ourlin, J. C., Clair, P., Gervot, L, Lesca, P, Domergue, J., Joyeux, H., Fourtanier, G., and Maurel, P (1994) Omeprazole and lansoprazole are mixed inducers of CYPlA and CYP3A in human hepato-cytes in primary culture. J Pharmacol Exp Ther 269, 384–392
Park, G. R., Pichard, L, Tinel, M, Larroque, C, Elston, A, Domergue, J., Detxonne, B., and Maurel, P. (1994) What changes drug metabolism in crtttcally ill patients? Two preliminary studies in isolated human hepatocytes. Anaesthesia 49, 188–191
Maurel, P (1996) The CYP3A family, in Cytochromes P450 Metabolic and Toxz-cological Aspects (ei]_Ioannides, C, ed.), CRC, Boca Raton, FL, pp 241–270
Wright, M. C., Maurel, P, and Paine, A. J. (1996) Induction of cytochrome P4503A by metyrapone in human hepatocyte in culture Human Exp Toxicol 15, 203,204
Greuet, J, Pichard, L, Bonfils, C, Domergue, J, and Maurel, P. (1996) The fetal specific gene CYP3A7 is inducible by rifampicin in adult human hepatocytes in primary culture. Biochem Biophys Res Commun 225, 689–694
Fabre, G., Bourrie, M., Marti, E., Fabre, I., Roque, C., Saint Aubert, B, Joyeux, H, Maurel, P, Berger, Y., and Cano, J. P (1990) Inducibility of both cytochrome P450 and UDP-glucuronosyl-transferase isozymes in primary cultures of human hepatocytes. Biochem Pharmacol (Life Sci. Adv) 9, 79–91
Abid, A, Sabolovic, N., Batt, A. M., Siest, G., Maurel, P., Gmllemin, F., and Magdalou, J (1994) Induction of UDP-glucuronosyltransferases and cytochromes P450 in human hepatocytes. Cell. Pharmacol. 1, 257–262.
Ptchard, L, Fabre, I, Fabre, G, Domergue, J, Saint Aubert, B., Mourad, G, and Maurel, P (1990) Cyclosporin A drug interactions. screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes. Drug Metab Dispos 18, 595–606
Maurice, M., Pichard, L., DauJat, M., Fabre, I, Joyeux, H., Domergue, J, and Maurel, P (1992) Effects of imidazole dertvatives on cytochromes P450 from human hepatocytes in primary culture FASEB J 6, 752–758
Muntane-Relat, J, Ourlin, J C, Domergue, J., and Maurel, P. (1995) Differential effects of cytokines on the inducible expression of CYPA1, CYP1A2, and1 CYP3A4 in human hepatocytes in primary culture. Hepatology 22, 1143–1153
Blanc, P., Etienne, H., Daujat, M., Fabre, I., Pichard, L., Domergue, J., Joyeux, H, Fourtanier, G., and Maurel, P (1991) Antiproliferative effect of FK506 and cyclosporin on adult human hepatocytes in culture. Transplant Proc 23, 2821–2824
Blanc, P., Etienne, H., Daujat, M., Fabre, I., Zindy, F, Domergue, J, Astre, C., SaintAubert, B, Michel, H, and Maurel, P (1992) Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor a, and human serum. Gastroenterology 102, 1340–1350.
Blanc, P., Liautard, J., Greuet, J., Daures, J. P., Fabre, J M., Larrey, D, Michel, H., and Maurel, P. (1995) Effect of anti-ulcer drugs on DNA synthesis in adult normal human hepatocytes in culture. Hepatologv 22, 814–819.
Ferrini, J. B, Pichard, L., Domergue, J., and Maurel, P. (1997) Long-term pri-mary cultures of adult human hepatocytes. Chemico-Biol Inter 107, 3l–45
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Ferrini, J.B., Ourlin, JC., Pichard, L., Fabre, G., Maurel, P. (1998). Human Hepatocyte Culture. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology™, vol 107. Humana Press. https://doi.org/10.1385/0-89603-519-0:341
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DOI: https://doi.org/10.1385/0-89603-519-0:341
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