Culture Systems for Hepatocytes for Use in Toxicology and Differentiation Studies

  • Gennady Ilyin
  • Anne Corlu
  • Pascal Loyer
  • Christiane Guguen-Guillouzo
Part of the Springer Lab Manual book series (SLM)


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.


Hepatocyte Growth Factor Human Hepatocyte Hepatocyte Culture Liver Specific Function Hydrocortisone Hemisuccinate 
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  1. 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 45:1583–1591PubMedCrossRefGoogle Scholar
  2. 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 244:233–235Google Scholar
  3. 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 4:839–842PubMedCrossRefGoogle Scholar
  4. Bissell DM, Arenson DM, Maher JJ, Roll FJ (1987) Support of cultured hepatocytes by a laminin-rich gel. J Clin Invest 79:801–812PubMedCrossRefGoogle Scholar
  5. 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 132:1133–1149PubMedCrossRefGoogle Scholar
  6. 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 289–309Google Scholar
  7. 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 236:263–268Google Scholar
  8. Caron JM (1990) Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol Cell Biol 10:1239–1243PubMedGoogle Scholar
  9. Clayton DF, Darnell JE (1983) Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol 3:1552–1561PubMedGoogle Scholar
  10. 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 4:373–380PubMedCrossRefGoogle Scholar
  11. 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 115:505–515PubMedCrossRefGoogle Scholar
  12. 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 11:147–153PubMedCrossRefGoogle Scholar
  13. 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 78:279–287PubMedCrossRefGoogle Scholar
  14. Edelman GM (1987) CAMs and Igs: cell adhesion and the evolutionary origins of imunity. Immunol Rev 100: 11–45PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 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 3:255–262PubMedGoogle Scholar
  17. 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 205:847–852PubMedCrossRefGoogle Scholar
  18. 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 204:237–241PubMedGoogle Scholar
  19. 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 4:2487–2491PubMedGoogle Scholar
  20. 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 228:238–245PubMedCrossRefGoogle Scholar
  21. Guengerich FP, Liebler DC (1985) Enzymatic activation of chemicals to toxic metabolites. CRC Crit Rev Toxicol 14:259–307CrossRefGoogle Scholar
  22. 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 143:47–54PubMedCrossRefGoogle Scholar
  23. 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 17:369–377PubMedCrossRefGoogle Scholar
  24. 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 12:1329–1336PubMedCrossRefGoogle Scholar
  25. 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 50:7460–7467PubMedGoogle Scholar
  26. 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 30:445–600PubMedCrossRefGoogle Scholar
  27. Houssaint E (1980) Differentiation of the mouse hepatic primordium. I. An analysis of tissue interactions in hepatocyte differentiation. Cell Diff 9:269–279CrossRefGoogle Scholar
  28. 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 187–208Google Scholar
  29. 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 105:2877–2885PubMedCrossRefGoogle Scholar
  30. 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 147:274–280PubMedCrossRefGoogle Scholar
  31. Kuri-Harcuch W, Mendoza-Figueroa T (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41:148–157PubMedCrossRefGoogle Scholar
  32. 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 143–152Google Scholar
  33. Lee J, Morgan JR, Tompkins RG, Yarmush ML (1993) Proline-mediated enhancement of hepatocyte function in a collagen gel sandwich culture configuration. FASEB J 7:586–591PubMedGoogle Scholar
  34. 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 244:98–106PubMedCrossRefGoogle Scholar
  35. Loeper J, Descatoire V, Maurice M et al. (1993) Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies. Gastroenterology 104:203–21PubMedGoogle Scholar
  36. Clément B (1993) Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro. Am J Pathol 143:538–544PubMedGoogle Scholar
  37. 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 271:11484–11492PubMedCrossRefGoogle Scholar
  38. 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 37–47CrossRefGoogle Scholar
  39. Mehendale HM, Roth RA, Gandolfi AE, Klaunig JE, Lemasters JJ, Curtis LR (1994) Novel mechanisms in chemically induced hepatotoxicity. FASEB J 8:1285–1295PubMedGoogle Scholar
  40. Miners JO, MacKenzie PI (1991) Drug glucuronidation in humans. Pharmacol Ther 51: 347–369PubMedCrossRefGoogle Scholar
  41. 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 13:21–30PubMedCrossRefGoogle Scholar
  42. 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 159:176–190PubMedCrossRefGoogle Scholar
  43. 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 14:1341–1352PubMedCrossRefGoogle Scholar
  44. Morin O, Norman C (1986) Long term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone. JCellPhys 129:103–110Google Scholar
  45. 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: 621–624Google Scholar
  46. 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 14:5858–5869PubMedGoogle Scholar
  47. 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 40:1701–1706PubMedCrossRefGoogle Scholar
  48. 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 46:805–814CrossRefGoogle Scholar
  49. 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 24:143–155PubMedCrossRefGoogle Scholar
  50. Seglen PO (1975) Preparation of isolated rat liver cells. Meth Cell Biol 13:29–83CrossRefGoogle Scholar
  51. 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 264:737–744PubMedGoogle Scholar
  52. 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 150:51–58PubMedGoogle Scholar
  53. 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 37:372–376PubMedGoogle Scholar
  54. Vickers AEM (1994) Use of human organ slices to evaluate the biotransformation and drug-induced side-effects of pharmaceuticals. Cell Biol Toxicol 10:407–414PubMedCrossRefGoogle Scholar
  55. 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 202:1–8PubMedCrossRefGoogle Scholar
  56. 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 35:519–525PubMedGoogle Scholar
  57. Williams GME, Weisburger EK, Weisburger JH (1971) Isolation and long-term cell culture of epithelial like cells from rat liver. Exp Cell Res 69:106–112PubMedCrossRefGoogle Scholar
  58. 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 43:2337–2343CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Gennady Ilyin
  • Anne Corlu
  • Pascal Loyer
  • Christiane Guguen-Guillouzo
    • 1
  1. 1.Hôpital PontchaillouINSERM U 49RennesFrance

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