Skip to main content

In Vitro Systems for Hepatotoxicity Testing

  • Protocol
  • First Online:
In Vitro Toxicology Systems

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Hepatotoxicity is the most frequent reason of drug withdrawal from the market. Therefore, hepatocyte in vitro systems that predict human hepatotoxicity are of high importance. Although some progress has been achieved in predicting toxicity formation of major metabolites and enzyme induction (Hewitt et al., Drug Metab Rev 39:159–234, 2007; Hengstler et al., Chem Biol Interact 125:51–73, 2000) it is still difficult to reliably predict idiosyncratic drug-induced liver injury (iDILI), a particularly worrying form of hepatotoxicity that can arise from many commonly prescribed drugs (Godoy et al., Arch Toxicol 87:1315–1530, 2013; Amacher, Expert Opin Drug Metab Toxicol 8:335–347, 2012). This chapter describes currently available hepatocyte in vitro systems and their possibilities as well as limitations in studying hepatotoxicity and ADME.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM, Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39(1):159–234

    Article  CAS  PubMed  Google Scholar 

  2. Hengstler JG, Ringel M, Biefang K, Hammel S, Milbert U, Gerl M, Klebach M, Diener B, Platt KL, Bottger T, Steinberg P, Oesch F (2000) Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction. Chem Biol Interact 125(1):51–73

    Article  CAS  PubMed  Google Scholar 

  3. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Bottger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gomez-Lechon MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Haussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhutter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, Lecluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stober R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Amacher DE (2012) The primary role of hepatic metabolism in idiosyncratic drug-induced liver injury. Expert Opin Drug Metab Toxicol 8(3):335–347

    Article  CAS  PubMed  Google Scholar 

  5. Hoehme S, Brulport M, Bauer A, Bedawy E, Schormann W, Hermes M, Puppe V, Gebhardt R, Zellmer S, Schwarz M, Bockamp E, Timmel T, Hengstler JG, Drasdo D (2010) Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration. Proc Natl Acad Sci U S A 107(23):10371–10376

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Ding BS, Nolan DJ, Butler JM, James D, Babazadeh AO, Rosenwaks Z, Mittal V, Kobayashi H, Shido K, Lyden D, Sato TN, Rabbany SY, Rafii S (2010) Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468(7321):310–315

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Goldin RD, Ratnayaka ID, Breach CS, Brown IN, Wickramasinghe SN (1996) Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. J Pathol 179(4): 432–435

    Article  CAS  PubMed  Google Scholar 

  8. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3(1):23–35

    Article  CAS  PubMed  Google Scholar 

  9. Mantovani A, Cassatella MA, Costantini C, Jaillon S (2011) Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol 11(8):519–531

    Article  CAS  PubMed  Google Scholar 

  10. Notas G, Kisseleva T, Brenner D (2009) NK and NKT cells in liver injury and fibrosis. Clin Immunol 130(1):16–26

    Article  CAS  PubMed  Google Scholar 

  11. Ochi M, Ohdan H, Mitsuta H, Onoe T, Tokita D, Hara H, Ishiyama K, Zhou W, Tanaka Y, Asahara T (2004) Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice. Hepatology 39(5):1321–1331

    Article  CAS  PubMed  Google Scholar 

  12. Tuschl G, Hrach J, Walter Y, Hewitt PG, Mueller SO (2009) Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: a valuable model for in vitro toxicity and drug-drug interaction studies. Chem Biol Interact 181(1): 124–137

    Article  CAS  PubMed  Google Scholar 

  13. Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Muller A, Tuschl G, Mueller SO, Dooley S (2009) Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 49(6): 2031–2043

    Article  CAS  PubMed  Google Scholar 

  14. Reder-Hilz B, Ullrich M, Ringel M, Hewitt N, Utesch D, Oesch F, Hengstler JG (2004) Metabolism of propafenone and verapamil by cryopreserved human, rat, mouse and dog hepatocytes: comparison with metabolism in vivo. Naunyn Schmiedebergs Arch Pharmacol 369(4):408–417

    Article  CAS  PubMed  Google Scholar 

  15. Carmo H, Hengstler JG, de Boer D, Ringel M, Remiao F, Carvalho F, Fernandes E, dos Reys LA, Oesch F, de Lourdes Bastos M (2005) Metabolic pathways of 4-bromo-2,5-dimethoxyphenethylamine (2C-B): analysis of phase I metabolism with hepatocytes of six species including human. Toxicology 206(1): 75–89

    Article  CAS  PubMed  Google Scholar 

  16. Ullrich A, Berg C, Hengstler JG, Runge D (2007) Use of a standardised and validated long-term human hepatocyte culture system for repetitive analyses of drugs: repeated administrations of acetaminophen reduces albumin and urea secretion. ALTEX 24(1): 35–40

    PubMed  Google Scholar 

  17. Ullrich A, Stolz DB, Ellis EC, Strom SC, Michalopoulos GK, Hengstler JG, Runge D (2009) Long term cultures of primary human hepatocytes as an alternative to drug testing in animals. ALTEX 26(4):295–302

    PubMed  Google Scholar 

  18. Knobeloch D, Ehnert S, Schyschka L, Buchler P, Schoenberg M, Kleeff J, Thasler WE, Nussler NC, Godoy P, Hengstler J, Nussler AK (2012) Human hepatocytes: isolation, culture, and quality procedures. Methods Mol Biol 806:99–120

    Article  CAS  PubMed  Google Scholar 

  19. Klingmuller U, Bauer A, Bohl S, Nickel PJ, Breitkopf K, Dooley S, Zellmer S, Kern C, Merfort I, Sparna T, Donauer J, Walz G, Geyer M, Kreutz C, Hermes M, Gotschel F, Hecht A, Walter D, Egger L, Neubert K, Borner C, Brulport M, Schormann W, Sauer C, Baumann F, Preiss R, MacNelly S, Godoy P, Wiercinska E, Ciuclan L, Edelmann J, Zeilinger K, Heinrich M, Zanger UM, Gebhardt R, Maiwald T, Heinrich R, Timmer J, von Weizsacker F, Hengstler JG (2006) Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways. Syst Biol (Stevenage) 153(6):433–447

    Article  CAS  Google Scholar 

  20. Peshwa MV, Wu FJ, Sharp HL, Cerra FB, Hu WS (1996) Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids. In Vitro Cell Dev Biol Anim 32(4): 197–203

    Article  CAS  PubMed  Google Scholar 

  21. Tzanakakis ES, Hansen LK, Hu WS (2001) The role of actin filaments and microtubules in hepatocyte spheroid self-assembly. Cell Motil Cytoskeleton 48(3):175–189

    Article  CAS  PubMed  Google Scholar 

  22. Chang TT, Hughes-Fulford M (2009) Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes. Tissue Eng Part A 15(3):559–567

    Article  CAS  PubMed  Google Scholar 

  23. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83(2):173–180

    Article  CAS  PubMed  Google Scholar 

  24. Landry J, Bernier D, Ouellet C, Goyette R, Marceau N (1985) Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J Cell Biol 101(3): 914–923

    Article  CAS  PubMed  Google Scholar 

  25. Kim BS, Park IK, Hoshiba T, Jiang HL, Choi YJ, Akaike T, Cho CS (2011) Design of artificial extracellular matrices for tissue engineering. Prog Polym Sci 36(2):239–268

    Article  Google Scholar 

  26. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, Martin GR (1986) Basement membrane complexes with biological activity. Biochemistry 25(2): 312–318

    Article  CAS  PubMed  Google Scholar 

  27. Ranucci CS, Kumar A, Batra SP, Moghe PV (2000) Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis. Biomaterials 21(8):783–793

    Article  CAS  PubMed  Google Scholar 

  28. Rowley JA, Madlambayan G, Mooney DJ (1999) Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20(1): 45–53

    Article  CAS  PubMed  Google Scholar 

  29. Semino CE, Merok JR, Crane GG, Panagiotakos G, Zhang S (2003) Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation 71(4–5):262–270

    Article  CAS  PubMed  Google Scholar 

  30. Hou YT, Ijima H, Matsumoto S, Kubo T, Takei T, Sakai S, Kawakami K (2010) Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes. J Biosci Bioeng 110(2):208–216

    Article  CAS  PubMed  Google Scholar 

  31. Ringel M, von Mach MA, Santos R, Feilen PJ, Brulport M, Hermes M, Bauer AW, Schormann W, Tanner B, Schon MR, Oesch F, Hengstler JG (2005) Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction. Toxicology 206(1): 153–167

    Article  CAS  PubMed  Google Scholar 

  32. Seo SJ, Choi YJ, Akaike T, Higuchi A, Cho CS (2006) Alginate/galactosylated chitosan/heparin scaffold as a new synthetic extracellular matrix for hepatocytes. Tissue Eng 12(1): 33–44

    Article  CAS  PubMed  Google Scholar 

  33. Sachlos E, Czernuszka JT (2003) Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 5:29–39, discussion 39–40

    CAS  PubMed  Google Scholar 

  34. Putnam AJ, Mooney DJ (1996) Tissue engineering using synthetic extracellular matrices. Nat Med 2(7):824–826

    Article  CAS  PubMed  Google Scholar 

  35. Chung TW, Yang J, Akaike T, Cho KY, Nah JW, Kim SI, Cho CS (2002) Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment. Biomaterials 23(14): 2827–2834

    Article  CAS  PubMed  Google Scholar 

  36. Hollister SJ (2005) Porous scaffold design for tissue engineering. Nat Mater 4(7):518–524

    Article  CAS  PubMed  Google Scholar 

  37. Cho CS, Goto M, Kobayashi A, Kobayashi K, Akaike T (1996) Effect of ligand orientation on hepatocyte attachment onto the poly(N-p-vinylbenzyl-o-beta-D-galactopyranosyl-D-gluconamide) as a model ligand of asialoglycoprotein. J Biomater Sci Polym Ed 7(12):1097–1104

    Article  CAS  PubMed  Google Scholar 

  38. Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, Milwid J, Kobayashi N, Tilles A, Berthiaume F, Hertl M, Nahmias Y, Yarmush ML, Uygun K (2010) Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 16(7): 814–820

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Zeng G, Awan F, Otruba W, Muller P, Apte U, Tan X, Gandhi C, Demetris AJ, Monga SP (2007) Wnt’er in liver: expression of Wnt and frizzled genes in mouse. Hepatology 45(1): 195–204

    Article  CAS  PubMed  Google Scholar 

  40. Krebs HA (1933) Untersuchungen über den Stoffwechsel der Aminosäuren im Tierkörper. Hoppe-Seyl Z 217:190

    Article  Google Scholar 

  41. Warburg O (1923) Versuche an u¨berlebendem Karzinomgewebe. Biochem Z 142:317

    CAS  Google Scholar 

  42. Krumdieck CL, dos Santos JE, Ho KJ (1980) A new instrument for the rapid preparation of tissue slices. Anal Biochem 104(1):118–123

    Article  CAS  PubMed  Google Scholar 

  43. Worboys PD, Bradbury A, Houston JB (1997) Kinetics of drug metabolism in rat liver slices. III. Relationship between metabolic clearance and slice uptake rate. Drug Metab Dispos 25(4):460–467

    CAS  PubMed  Google Scholar 

  44. Olinga P, Merema MT, Meijer DKF, Slooff MJH, Groothuis GMM (1993) Human liver slices express the same lidocaine biotransformation rate as isolated human hepatocytes. ATLA 21:466–468

    Google Scholar 

  45. Martin H, Sarsat JP, Lerche-Langrand C, Housset C, Balladur P, Toutain H, Albaladejo V (2002) Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension. Cell Biol Toxicol 18(2):73–85

    Article  CAS  PubMed  Google Scholar 

  46. de Graaf IA, de Kanter R, de Jager MH, Camacho R, Langenkamp E, van de Kerkhof EG, Groothuis GM (2006) Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction. Drug Metab Dispos 34(4):591–599

    Article  PubMed  Google Scholar 

  47. Olinga P, Groen K, Hof IH, De Kanter R, Koster HJ, Leeman WR, Rutten AA, Van Twillert K, Groothuis GM (1997) Comparison of five incubation systems for rat liver slices using functional and viability parameters. J Pharmacol Toxicol Methods 38(2):59–69

    Article  CAS  PubMed  Google Scholar 

  48. Olinga P, Elferink MG, Draaisma AL, Merema MT, Castell JV, Perez G, Groothuis GM (2008) Coordinated induction of drug transporters and phase I and II metabolism in human liver slices. Eur J Pharm Sci 33(4–5): 380–389

    Article  CAS  PubMed  Google Scholar 

  49. de Graaf IA, Olinga P, de Jager MH, Merema MT, de Kanter R, van de Kerkhof EG, Groothuis GM (2010) Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc 5(9):1540–1551

    Article  PubMed  Google Scholar 

  50. Klassen LW, Thiele GM, Duryee MJ, Schaffert CS, DeVeney AL, Hunter CD, Olinga P, Tuma DJ (2008) An in vitro method of alcoholic liver injury using precision-cut liver slices from rats. Biochem Pharmacol 76(3):426–436

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Vickers AE, Saulnier M, Cruz E, Merema MT, Rose K, Bentley P, Olinga P (2004) Organ slice viability extended for pathway characterization: an in vitro model to investigate fibrosis. Toxicol Sci 82(2):534–544

    Article  CAS  PubMed  Google Scholar 

  52. Miller LL, Bly CG, Watson ML, Bale WF (1951) The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14. J Exp Med 94(5): 431–453

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Sies H (1978) The use of perfusion of liver and other organs for the study of microsomal electron-transport and cytochrome P-450 systems. Methods Enzymol 52:48–59

    Article  CAS  PubMed  Google Scholar 

  54. Haussinger D (1987) Isolated perfused rat liver: an experimental model for studies on ammonium and amino acid metabolism. Infusionsther Klin Ernahr 14(4):174–178

    CAS  PubMed  Google Scholar 

  55. vom Dahl S, Haussinger D (1997) Experimental methods in hepatology. Guidelines of the German Association for the Study of the Liver (GASL). Liver perfusion—technique and applications. Z Gastroenterol 35(3):221–226

    Google Scholar 

  56. Tsuruga Y, Kiyono T, Matsushita M, Takahashi T, Kasai H, Matsumoto S, Todo S (2008) Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy. Cell Transplant 17(9): 1083–1094

    Article  PubMed  Google Scholar 

  57. Wege H, Le HT, Chui MS, Liu L, Wu J, Giri R, Malhi H, Sappal BS, Kumaran V, Gupta S, Zern MA (2003) Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 124(2):432–444

    Article  CAS  PubMed  Google Scholar 

  58. Kobayashi N, Noguchi H, Westerman KA, Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Leboulch P, Tanaka N (2001) Cre/loxP-based reversible immortalization of human hepatocytes. Cell Transplant 10(4–5):383–386

    CAS  PubMed  Google Scholar 

  59. Hart SN, Li Y, Nakamoto K, Subileau EA, Steen D, Zhong XB (2010) A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues. Drug Metab Dispos 38(6):988–994

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jan G. Hengstler .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Hengstler, J.G., Hammad, S., Ghallab, A., Reif, R., Godoy, P. (2014). In Vitro Systems for Hepatotoxicity Testing. In: Bal-Price, A., Jennings, P. (eds) In Vitro Toxicology Systems. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0521-8_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0521-8_2

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0520-1

  • Online ISBN: 978-1-4939-0521-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics