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Culture Systems for Hepatocytes for Use in Toxicology and Differentiation Studies

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Animal Cell Culture Techniques

Part of the book series: Springer Lab Manual ((SLM))

Abstract

Owing to the high level of biotransformation enzymes in the liver, hepatocyte cultures are potentially valuable in vitro test systems for determining the metabolic profiles of drugs as well as for measuring the activities and substrate specificities of the various hepatic enzyme systems involved in their biotransformation. Hepatocyte cultures are also of major interest as they can be used to determine the potential toxicity of newly formed metabolites. In this chapter, our aim is to describe both in vitro assays that can readily be adopted by several laboratories and prediction models that can easily be interpreted. To understand the benefits and, more importantly, the limitations of the different hepatocyte models used in toxicology and drug metabolism, it is necessary to be acquainted with the main features of those systems currently in use. This chapter updates the discussion of different model systems of hepatocyte primary cultures and examines one of the main functions of the cells, i.e., drug metabolism, in relation to proliferation, differentiation status or apoptotic response.

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References

  • Akrawi M, Rogiers V, Vandenberghe Y, Palmer CNA, Vercruysse A, Shephard EA, Phillips IR (1993) Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P-450-mediated mono-oxygenase. Biochem Pharmacol 45:1583–1591

    Article  PubMed  CAS  Google Scholar 

  • Ballet F, Chretien Y, Rey C, Poupon R (1988) Differential response of normal and cirrhotic liver to vasoactive agents. A study in the isolated perfused rat liver. J Pharmacol Exp Ther 244:233–235

    Google Scholar 

  • Bégué JM, Guguen-Guillouzo C, Pasdeloup N, Guillouzo A (1984) Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology 4:839–842

    Article  PubMed  Google Scholar 

  • Bissell DM, Arenson DM, Maher JJ, Roll FJ (1987) Support of cultured hepatocytes by a laminin-rich gel. J Clin Invest 79:801–812

    Article  PubMed  CAS  Google Scholar 

  • Block GD, Locker J, Bowen WC, Petersen BE, Katyal S, Strom SC, Riley T, Howard TA, Michalopoulos GK (1996) Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGFα in a chemically defined (HGM) medium. J Cell Biol 132:1133–1149

    Article  PubMed  CAS  Google Scholar 

  • Bock KW (1995) Human UDP-glucuronosyl transferases: classification and properties of isozymes. In: Paifici GM, Francchia GN (eds) Advances in drug metabolism in man. European Commission, Bruxelles, pp 289–309

    Google Scholar 

  • Bradford BU, Marotto M, Lemasters JJ, Thurman RG (1986) New, simple models to evaluate zone-specific damage due to hypoxia in the perfused rat liver: time course and effect of nutritional state. J Pharm Exp Ther 236:263–268

    CAS  Google Scholar 

  • Caron JM (1990) Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol 10:1239–1243

    PubMed  CAS  Google Scholar 

  • Clayton DF, Darnell JE (1983) Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol 3:1552–1561

    PubMed  CAS  Google Scholar 

  • Clement B, Guguen-Guillouzo C, Campion JP, Glaise D, Bourel M, Guillouzo A (1984) Long-term co-cultures of adult human hepatocytes with rat liver epithelial cells: modulation of active albumin secretion and accumulation of extracellular material. Hepatology 4:373–380

    Article  PubMed  CAS  Google Scholar 

  • Corlu A, Kneip B, Lhadi C, Leray G, Glaise D, Baffet G, Bourel D, Guguen-Guillouzo C (1991) A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes. J Cell Biol 115:505–515

    Article  PubMed  CAS  Google Scholar 

  • de Sousa G, Florence N, Valles B, Coassolo P, Rahmani R (1995) Relationship between in vitro and in vivo biotransformation of drugs in humans and animals: pharmacotoxicological consequences. Cell Biol Toxicol 11:147–153

    Article  PubMed  Google Scholar 

  • Decad GM, Hsieh DPH, Byard JL (1977) Maintenance of cytochrome P-450 and metabolism of aflatoxin B1 in primary hepatocyte cultures. Biochem. Biophys Res Commun 78:279–287

    Article  PubMed  CAS  Google Scholar 

  • Edelman GM (1987) CAMs and Igs: cell adhesion and the evolutionary origins of imunity. Immunol Rev 100: 11–45

    Article  PubMed  CAS  Google Scholar 

  • Enat R, Jefferson DM, Ruiz-Opazo N, Gatmaitan Z, Leinwand L, Reid LM (1984) Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc Natl Acad Sci USA 81:1411–1415.

    Article  PubMed  CAS  Google Scholar 

  • Etienne PL, Baffet G, Desvergne B, Boisnard-Rissel M, Glaise D, Guguen-Guillouzo C (1988) Transient expression of c-fos and constant expression of c-myc in freshly isolated and cultured normal adult rat hepatocytes. Oncogene Res 3:255–262

    PubMed  CAS  Google Scholar 

  • Fardel O, Ratanasavanh D, Loyer P, Ketterer B, Guillouzo A (1992) Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture. Eur J Biochem 205:847–852

    Article  PubMed  CAS  Google Scholar 

  • Fausto N, Lemire JM, Shiojiri N (1993) Cell lineages in hepatic development and the identification of progenitor cells in normal and injured liver. Proc Soc Exp Biol Med 204:237–241

    PubMed  CAS  Google Scholar 

  • Fraslin JM, Kneip B, Vaulont S, Glaise D, Munnich A, Guguen-Guillouzo C (1985) Dependence of hepatocyte specific gene expression on cell-cell interactions in primary culture. EMBO J 4:2487–2491

    PubMed  CAS  Google Scholar 

  • Gardner MJ, Fletcher K, Pogson CI, Strain AJ (1996) The mitogenic response to EGF of rat hepatocytes cultured on laminin-rich gels (EHS) is blocked downstream of receptor tyrosine-phosphorylation. Biochem Biophys Res Comm 228:238–245

    Article  PubMed  CAS  Google Scholar 

  • Guengerich FP, Liebler DC (1985) Enzymatic activation of chemicals to toxic metabolites. CRC Crit Rev Toxicol 14:259–307

    Article  CAS  Google Scholar 

  • Guguen-Guillouzo C, Clément B, Baffet G, Beaumont C, Morel-Chany E, Glaise D, Guillouzo A (1983) Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp Cell Res 143:47–54

    Article  PubMed  CAS  Google Scholar 

  • Guguen-Guillouzo C, Szajnert MF, Glaise D, Gregory C, Schapira F (1981) Isozyme differentiation of aldolase and pyruvate kinase in fetal, regenerating, preneoplastic and malignant rat hepatocytes during culture. In Vitro 17:369–377

    Article  PubMed  CAS  Google Scholar 

  • Guyomard C, Chesné C, Meunier B, Fautrel A, Clerc C, Morel F, Rissel M, Campion JP, Guillouzo A (1990) Primary culture of adult rat hepatocytes after 48 hour preservation of the liver with cold UW solution. Hepatology 12:1329–1336

    Article  PubMed  CAS  Google Scholar 

  • Hampton LL, Worland PJ, Yu B, Thorgeirsson SS, Huggett AC (1990) Expression of growth-related genes during tumor progression in v-raf-transformed rat liver epithelial cells. Cancer Res 50:7460–7467

    PubMed  CAS  Google Scholar 

  • Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: Regulation of GST and the contribution of the isozymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30:445–600

    Article  PubMed  CAS  Google Scholar 

  • Houssaint E (1980) Differentiation of the mouse hepatic primordium. I. An analysis of tissue interactions in hepatocyte differentiation. Cell Diff 9:269–279

    Article  CAS  Google Scholar 

  • Ichihara A, Nakamura T, Noda C, Tanaka K (1986) Control of enzyme expression deduced from studies on primary cultures of hepatocytes. In: Guillouzo A, Guguen-Guillouzo C (eds) Isolated and cultured hepatocytes. John Libbey Euro-text Ltd/INSERM, London, pp 187–208

    Google Scholar 

  • Isom H, Georgoff I, Salditt-Georgieff M, Darnell JE (1987) Persistence of liver-specific messenger RNA in cultured hepatocytes: different regulatory events for different genes. J Cell Biol 105:2877–2885

    Article  PubMed  CAS  Google Scholar 

  • Kost DP, Michalopoulos GK (1991) Effect of 2 % dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture. J Cell Physiol 147:274–280

    Article  PubMed  CAS  Google Scholar 

  • Kuri-Harcuch W, Mendoza-Figueroa T (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41:148–157

    Article  PubMed  CAS  Google Scholar 

  • Larrey D, Pessayre D (1988) Genetic factors in hepatotoxicity. In: Guillouzo A (ed) Liver cells and drugs. Les Editions INSERM and John Libbey Eurotext, Paris, pp 143–152

    Google Scholar 

  • Lee J, Morgan JR, Tompkins RG, Yarmush ML (1993) Proline-mediated enhancement of hepatocyte function in a collagen gel sandwich culture configuration. FASEB J 7:586–591

    PubMed  CAS  Google Scholar 

  • Lerche C, Fautrel A, Shaw PM, Glaise D, Ballet F, Guillouzo A, Corcos L (1997) Regulation of the major detoxication functions by phenobarbital and 3-methyl-cholanthrene in co-cultures of rat hepatocytes and liver epithelial cells. Eur J Biochem 244:98–106

    Article  PubMed  CAS  Google Scholar 

  • Loeper J, Descatoire V, Maurice M et al. (1993) Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies. Gastroenterology 104:203–21

    PubMed  CAS  Google Scholar 

  • Clément B (1993) Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro. Am J Pathol 143:538–544

    PubMed  Google Scholar 

  • Loyer P, Cariou S, Glaise D, Bilodeau M, Baffet G, Guguen-Guillouzo C (1996) Growth factor-dependence of entry and progression through Gl and S phases of adult rat hepatocytes in vitro. J Biol Chem 271:11484–11492

    Article  PubMed  CAS  Google Scholar 

  • Loyer P, Ilyin G, Cariou S, Glaise D, Corlu A, Guguen-Guillouzo C (1996) Progression through Gl and S phases of adult rat hepatocytes. In: Meijer L, Guidet S, Vogel L (eds) Progress in cell cycle research, vol 2. Plenum Press, New York, pp 37–47

    Chapter  Google Scholar 

  • Mehendale HM, Roth RA, Gandolfi AE, Klaunig JE, Lemasters JJ, Curtis LR (1994) Novel mechanisms in chemically induced hepatotoxicity. FASEB J 8:1285–1295

    PubMed  CAS  Google Scholar 

  • Miners JO, MacKenzie PI (1991) Drug glucuronidation in humans. Pharmacol Ther 51: 347–369

    Article  PubMed  CAS  Google Scholar 

  • Mitaka T, Sattler CA, Sattler GL, Sargent LM, Pitot HC (1991) Miltiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology 13:21–30

    Article  PubMed  CAS  Google Scholar 

  • Miyazaki M, Handa Y, Oda M, Yabe T, Miyano K, Sato J (1985) Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture. Exp Cell Res 159:176–190

    Article  PubMed  CAS  Google Scholar 

  • Morel-Chany E, Guillouzo C, Trincal G, Szajnert MF (1978) “Spontaneous” neoplastic transformation in vitro of epithelial cell strains of rat liver: cytology, growth and enzymatic activities. Eur J Cancer 14:1341–1352

    Article  PubMed  CAS  Google Scholar 

  • Morin O, Norman C (1986) Long term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone. JCellPhys 129:103–110

    CAS  Google Scholar 

  • Padgham CRW, Paine AJ (1993) Altered expression of cytochrome P-450 mRNAs, and potentially of other transcripts encoding key hepatic functions, are triggered during the isolation of rat hepatocytes. Biochem J, 28: 621–624

    Google Scholar 

  • Rana A, Mischoulon D, Xie Y, Bucher NL, Farmer SR (1994) Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP a and immediate-early growth response transcription factors. Mol Cell Biol 14:5858–5869

    PubMed  CAS  Google Scholar 

  • Rogiers V, Vandenberghe Y, Callaerts A, Verleye G, Cornet M, Mertens K, Sonk W, Vercruysse A (1990) Phase I and phase II xenobiotic biotransformation in cultures and cocultures of adult rat hepatocytes. Biochem Pharmacol 40:1701–1706

    Article  PubMed  CAS  Google Scholar 

  • Saad B, Scholl FA, Thomas H, Schwalder H, Streit V, Waechter F, Maier P (1993) Crude liver membrane fractions and extracellular matrix components as substrata regulate differentially the preservation and inducibility of cytochrome P-450 isoenzymes in cultured rat hepatocytes. Eur J Biochem 46:805–814

    Article  Google Scholar 

  • Sandker GW, Vos RM, Delbressine LP, Slooff MJ, Meijer DK, Groothuis GM (1994) Metabolism of three pharmacologically active drugs in isolated human and rat hepatocytes: analysis of interspecies variability and comparison with metabolism in vivo. Xenobiotica 24:143–155

    Article  PubMed  CAS  Google Scholar 

  • Seglen PO (1975) Preparation of isolated rat liver cells. Meth Cell Biol 13:29–83

    Article  Google Scholar 

  • Steinberg P, Schramm H, Schladt L, Robertson LW, Thomas H, Oesch F (1989) The distribution, induction and isoenzyme profile of glutathione -transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells. Biochem J 264:737–744

    PubMed  CAS  Google Scholar 

  • Theret N, Musso O, L’Helgoualc’h A, Clément B (1997) Activation of matrix metaloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes. Am J Pathol 150:51–58

    PubMed  CAS  Google Scholar 

  • Vandenberghe Y, Morel F, Pemble S, Taylor JB, Rogiers V, Ratanasavanh D, Vercruysse A, Ketterer B, Guillouzo A (1990) Changes in expression of mRNA coding for glutathione S-transferase subunits 1–2 and 7 in cultured rat hepatocytes. Mol Pharmacol 37:372–376

    PubMed  CAS  Google Scholar 

  • Vickers AEM (1994) Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals. Cell Biol Toxicol 10:407–414

    Article  PubMed  CAS  Google Scholar 

  • Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH (1992) Identification of multiple active growth factors in basement membrane matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 202:1–8

    Article  PubMed  CAS  Google Scholar 

  • Waxman DJ, Morrissey JJ, Leblanc GA (1989) Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase. Mol Pharmacol 35:519–525

    PubMed  CAS  Google Scholar 

  • Williams GME, Weisburger EK, Weisburger JH (1971) Isolation and long-term cell culture of epithelial like cells from rat liver. Exp Cell Res 69:106–112

    Article  PubMed  CAS  Google Scholar 

  • Wright MC, Paine AJ (1992) Evidence that the loss of rat liver cytochrome P-450 in vitro is not solely associated with the use of collagenase, the loss of cell-cell contacts and/or the absence of an extracellular matrix. Biochem Pharmacol 43:2337–2343

    Article  Google Scholar 

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Authors and Affiliations

  1. Hôpital Pontchaillou, INSERM U 49, 35033, Rennes, France

    Christiane Guguen-Guillouzo

Authors
  1. Gennady Ilyin
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  2. Anne Corlu
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  3. Pascal Loyer
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  4. Christiane Guguen-Guillouzo
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Corresponding author

Correspondence to Christiane Guguen-Guillouzo .

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Editors and Affiliations

  1. National Cell & Tissue Culture Center Bioresearch Ireland, Dublin City University, Glasnevin Dublin 9, Ireland

    Martin Clynes

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© 1998 Springer-Verlag Berlin Heidelberg

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Ilyin, G., Corlu, A., Loyer, P., Guguen-Guillouzo, C. (1998). Culture Systems for Hepatocytes for Use in Toxicology and Differentiation Studies. In: Clynes, M. (eds) Animal Cell Culture Techniques. Springer Lab Manual. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-80412-0_20

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  • DOI: https://doi.org/10.1007/978-3-642-80412-0_20

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-540-63008-1

  • Online ISBN: 978-3-642-80412-0

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