Skip to main content
SpringerLink
Log in

Primary hepatocytes and their cultures in liver apoptosis research

  • Review Article
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

APAF-1:

Apoptotic protease activating factor 1

Bcl-2:

B cell lymphoma 2

Caspase(s):

Cysteinyl aspartate-specific proteinase(s)

dATP:

Deoxyadenosine triphosphate

DiOC6 :

3,3-Dihexyloxacarbocyanine iodide

DMSO:

Dimethylsulphoxide

ECM:

Extracellular matrix

EGF:

Epidermal growth factor

ERK:

Extracellular signal-regulated kinase

HBV:

Hepatitis B virus

HBX:

Hepatitis B virus X protein

HCV:

Hepatitis C virus

HGF:

Hepatocyte growth factor

IAP:

Inhibitor of apoptosis

JNK:

c-Jun N-terminal kinase

LDH:

Lactate dehydrogenase

LPS:

Lipopolysaccharide

MAPK:

Mitogen-activated protein kinase

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

NF-κβ:

Nuclear factor-kappa beta

NO:

Nitric oxide

PI3K:

Phosphatidylinositol-3 kinase

SAPK:

Stress-activated protein kinase

TGFβ1:

Transforming growth factor β1

TNFα:

Tumour necrosis factor α

TRAIL:

Tumour necrosis factor α-related apoptosis-inducing ligand

TUNEL:

Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling

References

  • Abou-Elella AM, Siendones E, Padillo J, Montero JL, De la Mata M, Muntane Relat J (2002) Tumour necrosis factor-alpha and nitric oxide mediate apoptosis by d-galactosamine in a primary culture of rat hepatocytes: exacerbation of cell death by cocultured Kupffer cells. Can J Gastroenterol 16:791–799

    PubMed  Google Scholar 

  • Alpini G, Phillips JO, Vroman B, LaRusso NF (1994) Recent advances in the isolation of liver cells. Hepatology 20:494–514

    CAS  PubMed  Google Scholar 

  • Alvarez SD, Derfus AM, Schwartz MP, Bhatia SN, Sailor MJ (2009) The compatibility of hepatocytes with chemically modified porous silicon with reference to in vitro biosensors. Biomaterials 30:26–34

    CAS  PubMed Central  PubMed  Google Scholar 

  • Arterburn LM, Zurlo J, Yager JD, Overton RM, Heifetz AH (1995) A morphological study of differentiated hepatocytes in vitro. Hepatology 22:175–187

    CAS  PubMed  Google Scholar 

  • Arzberger S, Hosel M, Protzer U (2010) Apoptosis of hepatitis B virus-infected hepatocytes prevents release of infectious virus. J Virol 84:11994–12001

    CAS  PubMed Central  PubMed  Google Scholar 

  • Baier PK, Baumgartner U, Wolff-Vorbeck G, Hempel S, Hopt UT (2006) Hepatocyte proliferation and apoptosis in rat liver after liver injury. Hepatogastroenterology 53:747–752

    CAS  PubMed  Google Scholar 

  • Bailly-Maitre B, de Sousa G, Boulukos K, Gugenheim J, Rahmani R (2001) Dexamethasone inhibits spontaneous apoptosis in primary cultures of human and rat hepatocytes via Bcl-2 and Bcl-xL induction. Cell Death Differ 8:279–288

    CAS  PubMed  Google Scholar 

  • Bailly-Maitre B, de Sousa G, Zucchini N, Gugenheim J, Boulukos KE, Rahmani R (2002) Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone. Cell Death Differ 9:945–955

    CAS  PubMed  Google Scholar 

  • Banic B, Nipic D, Suput D, Milisav I (2011) DMSO modulates the pathway of apoptosis triggering. Cell Mol Biol Lett 16:328–341

    CAS  PubMed  Google Scholar 

  • Bellamy CO, Prost S, Wyllie AH, Harrison DJ (1997) UV but not gamma-irradiation induces specific transcriptional activity of p53 in primary hepatocytes. J Pathol 183:177–181

    CAS  PubMed  Google Scholar 

  • Benedetti A, Di Sario A, Svegliati Baroni G, Jezequel AM (1995) Transforming growth factor beta 1 increases the number of apoptotic bodies and decreases intracellular pH in isolated periportal and perivenular rat hepatocytes. Hepatology 22:1488–1498

    CAS  PubMed  Google Scholar 

  • Berry MN, Grivell AR, Grivell MB, Phillips JW (1997) Isolated hepatocytes-past, present and future. Cell Biol Toxicol 13:223–233

    CAS  PubMed  Google Scholar 

  • Bilodeau M, Tousignant J, Ethier C, Rocheleau B, Raymond VA, Lapointe R (2004) Anti-apoptotic effect of insulin on normal hepatocytes in vitro and in vivo. Apoptosis 9:609–617

    CAS  PubMed  Google Scholar 

  • Boer U, Fennekohl A, Puschel GP (2003) Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor alpha-induced apoptosis. J Hepatol 38:728–735

    CAS  PubMed  Google Scholar 

  • Bohnenberger S, Wagner B, Schmitz HJ, Schrenk D (2001) Inhibition of apoptosis in rat hepatocytes treated with ‘non-dioxin-like’ polychlorinated biphenyls. Carcinogenesis 22:1601–1606

    CAS  PubMed  Google Scholar 

  • Bresgen N, Ohlenschlager I, Wacht N, Afazel S, Ladurner G, Eckl PM (2008) Ferritin and FasL (CD95L) mediate density dependent apoptosis in primary rat hepatocytes. J Cell Physiol 217:800–808

    CAS  PubMed  Google Scholar 

  • Bresgen N, Rolinek R, Hochleitner E, Lottspeich F, Eckl PM (2004) Induction of apoptosis by a hepatocyte conditioned medium. J Cell Physiol 198:452–460

    CAS  PubMed  Google Scholar 

  • Carmona-Cuenca I, Roncero C, Sancho P, Caja L, Fausto N, Fernandez M, Fabregat I (2008) Upregulation of the NADPH oxidase NOX4 by TGF-beta in hepatocytes is required for its pro-apoptotic activity. J Hepatol 49:965–976

    CAS  PubMed  Google Scholar 

  • Choi BM, Pae HO, Kim YM, Chung HT (2003) Nitric oxide-mediated cytoprotection of hepatocytes from glucose deprivation-induced cytotoxicity: involvement of heme oxygenase-1. Hepatology 37:810–823

    CAS  PubMed  Google Scholar 

  • Christensen JG, Gonzales AJ, Cattley RC, Goldsworthy TL (1998) Regulation of apoptosis in mouse hepatocytes and alteration of apoptosis by nongenotoxic carcinogens. Cell Growth Differ 9:815–825

    CAS  PubMed  Google Scholar 

  • Clippinger AJ, Gearhart TL, Bouchard MJ (2009) Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore. J Virol 83:4718–4731

    CAS  PubMed Central  PubMed  Google Scholar 

  • Corazza N, Kassahn D, Jakob S, Badmann A, Brunner T (2009) TRAIL-induced apoptosis: between tumor therapy and immunopathology. Ann N Y Acad Sci 1171:50–58

    CAS  PubMed  Google Scholar 

  • Cosgrove BD, Cheng C, Pritchard JR, Stolz DB, Lauffenburger DA, Griffith LG (2008) An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha. Hepatology 48:276–288

    CAS  PubMed  Google Scholar 

  • Cosgrove BD, King BM, Hasan MA, Alexopoulos LG, Farazi PA, Hendriks BS, Griffith LG, Sorger PK, Tidor B, Xu JJ, Lauffenburger DA (2009) Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity. Toxicol Appl Pharmacol 237:317–330

    CAS  PubMed Central  PubMed  Google Scholar 

  • Crenesse D, Laurens M, Heurteaux C, Cursio R, Saint-Paul MC, Schmid-Alliana A, Gugenheim J (2003) Rat liver ischemia-reperfusion-induced apoptosis and necrosis are decreased by FK506 pretreatment. Eur J Pharmacol 473:177–184

    CAS  PubMed  Google Scholar 

  • Decrock E, Vinken M, De Vuyst E, Krysko DV, D’Herde K, Vanhaecke T, Vandenabeele P, Rogiers V, Leybaert L (2009) Connexin-related signaling in cell death: to live or let die? Cell Death Differ 16:524–536

    CAS  PubMed  Google Scholar 

  • Diao J, Khine AA, Sarangi F, Hsu E, Iorio C, Tibbles LA, Woodgett JR, Penninger J, Richardson CD (2001) X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway. J Biol Chem 276:8328–8340

    CAS  PubMed  Google Scholar 

  • Donato MT, Ponsoda X, O’Connor E, Castell JV, Gomez-Lechon MJ (2001) Role of endogenous nitric oxide in liver-specific functions and survival of cultured rat hepatocytes. Xenobiotica 31:249–264

    CAS  PubMed  Google Scholar 

  • Drudi Metalli V, Mancino MG, Mancino A, Torrice A, Gatto M, Attili AF, Alpini G, Alvaro D (2007) Bile salts regulate proliferation and apoptosis of liver cells by modulating the IGF1 system. Dig Liver Dis 39:654–662

    CAS  PubMed  Google Scholar 

  • Dunn JC, Tompkins RG, Yarmush ML (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog 7:237–245

    CAS  PubMed  Google Scholar 

  • Dunn JC, Yarmush ML, Koebe HG, Tompkins RG (1989) Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. Faseb J 3:174–177

    CAS  PubMed  Google Scholar 

  • Dvir-Ginzberg M, Elkayam T, Aflalo ED, Agbaria R, Cohen S (2004) Ultrastructural and functional investigations of adult hepatocyte spheroids during in vitro cultivation. Tissue Eng 10:1806–1817

    CAS  PubMed  Google Scholar 

  • Elaut G, Henkens T, Papeleu P, Snykers S, Vinken M, Vanhaecke T, Rogiers V (2006) Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab 7:629–660

    CAS  PubMed  Google Scholar 

  • Elaut G, Vanhaecke T, Heyden YV, Rogiers V (2005) Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium. Biochem Pharmacol 69:1829–1838

    CAS  PubMed  Google Scholar 

  • Ethier C, Raymond VA, Musallam L, Houle R, Bilodeau M (2003) Antiapoptotic effect of EGF on mouse hepatocytes associated with downregulation of proapoptotic Bid protein. Am J Physiol Gastrointest Liver Physiol 285:G298–G308

    CAS  PubMed  Google Scholar 

  • Fadeel B, Thorpe CJ, Yonehara S, Chiodi F (1997) Anti-Fas IgG1 antibodies recognizing the same epitope of Fas/APO-1 mediate different biological effects in vitro. Int Immunol 9:201–209

    CAS  PubMed  Google Scholar 

  • Feldmann G (1997) Liver apoptosis. J Hepatol 26(Suppl 2):1–11

    CAS  PubMed  Google Scholar 

  • Fladmark KE, Gjertsen BT, Doskeland SO, Vintermyr OK (1997) Fas/APO-1(CD95)-induced apoptosis of primary hepatocytes is inhibited by cAMP. Biochem Biophys Res Commun 232:20–25

    CAS  PubMed  Google Scholar 

  • Fu T, Blei AT, Takamura N, Lin T, Guo D, Li H, O’Gorman MR, Soriano HE (2004) Hypothermia inhibits Fas-mediated apoptosis of primary mouse hepatocytes in culture. Cell Transplant 13:667–676

    PubMed  Google Scholar 

  • Fu T, Guo D, Huang X, O’Gorman MR, Huang L, Crawford SE, Soriano HE (2001) Apoptosis occurs in isolated and banked primary mouse hepatocytes. Cell Transplant 10:59–66

    CAS  PubMed  Google Scholar 

  • Furukawa S, Usuda K, Fujieda Y, Tamura T, Miyamoto Y, Hayashi K, Ikeyama S, Goryo M, Okada K (2000) Apoptosis and cell proliferation in rat hepatocytes induced by barbiturates. J Vet Med Sci 62:23–28

    CAS  PubMed  Google Scholar 

  • Gilot D, Loyer P, Corlu A, Glaise D, Lagadic-Gossmann D, Atfi A, Morel F, Ichijo H, Guguen-Guillouzo C (2002) Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione S-transferase regulation. J Biol Chem 277:49220–49229

    CAS  PubMed  Google Scholar 

  • Gilot D, Serandour AL, Ilyin GP, Lagadic-Gossmann D, Loyer P, Corlu A, Coutant A, Baffet G, Peter ME, Fardel O, Guguen-Guillouzo C (2005) A role for caspase-8 and c-FLIPL in proliferation and cell-cycle progression of primary hepatocytes. Carcinogenesis 26:2086–2094

    CAS  PubMed  Google Scholar 

  • Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Müller 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 49:2031–2043

    CAS  PubMed  Google Scholar 

  • Gomez-Lechon MJ, O’Connor E, Castell JV, Jover R (2002) Sensitive markers used to identify compounds that trigger apoptosis in cultured hepatocytes. Toxicol Sci 65:299–308

    CAS  PubMed  Google Scholar 

  • Gomez-Quiroz LE, Factor VM, Kaposi-Novak P, Coulouarn C, Conner EA, Thorgeirsson SS (2008) Hepatocyte-specific c-Met deletion disrupts redox homeostasis and sensitizes to Fas-mediated apoptosis. J Biol Chem 283:14581–14589

    CAS  PubMed  Google Scholar 

  • Guguen-Guillouzo C, Clement 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

    CAS  PubMed  Google Scholar 

  • Guicciardi ME, Bronk SF, Werneburg NW, Yin XM, Gores GJ (2005) Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alpha-induced hepatocyte apoptosis. Gastroenterology 129:269–284

    CAS  PubMed  Google Scholar 

  • Guicciardi ME, Gores GJ (2005) Apoptosis: a mechanism of acute and chronic liver injury. Gut 54:1024–1033

    CAS  PubMed  Google Scholar 

  • Hamilton GA, Jolley SL, Gilbert D, Coon DJ, Barros S, LeCluyse EL (2001) Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell–cell interactions. Cell Tissue Res 306:85–99

    CAS  PubMed  Google Scholar 

  • Han D, Hanawa N, Saberi B, Kaplowitz N (2006) Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis. Free Radic Biol Med 41:627–639

    CAS  PubMed  Google Scholar 

  • Henkens T, Papeleu P, Elaut G, Vinken M, Rogiers V, Vanhaecke T (2007) Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes. Toxicol Appl Pharmacol 218:64–71

    CAS  PubMed  Google Scholar 

  • Hentze H, Latta M, Kunstle G, Dhakshinamoorthy S, Ng PY, Porter AG, Wendel A (2004) Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis. Hepatology 39:1311–1320

    CAS  PubMed  Google Scholar 

  • Hirschfield GM, Heathcote EJ, Gershwin ME (2010) Pathogenesis of cholestatic liver disease and therapeutic approaches. Gastroenterology 139:1481–1496

    CAS  PubMed  Google Scholar 

  • Hohenester S, Gates A, Wimmer R, Beuers U, Anwer MS, Rust C, Webster CR (2010) Phosphatidylinositol-3-kinase p110gamma contributes to bile salt-induced apoptosis in primary rat hepatocytes and human hepatoma cells. J Hepatol 53:918–926

    CAS  PubMed Central  PubMed  Google Scholar 

  • Holzer C, Maier P (1987) DNA and protein contents of hepatocytes in primary cultures monitored by flow cytometry: effect of phenobarbital and dimethylsulphoxide. Toxicol In Vitro 1:203–213

    CAS  PubMed  Google Scholar 

  • Hoshiba T, Nagahara H, Cho CS, Tagawa Y, Akaike T (2007) Primary hepatocyte survival on non-integrin-recognizable matrices without the activation of Akt signaling. Biomaterials 28:1093–1104

    CAS  PubMed  Google Scholar 

  • Hully JR, Chang L, Schwall RH, Widmer HR, Terrell TG, Gillett NA (1994) Induction of apoptosis in the murine liver with recombinant human activin A. Hepatology 20:854–862

    CAS  PubMed  Google Scholar 

  • Hussain SM, Frazier JM (2003) Involvement of apoptosis in hydrazine induced toxicity in rat primary hepatocytes. Toxicol In Vitro 17:343–355

    CAS  PubMed  Google Scholar 

  • Hynes J, Hill R, Papkovsky DB (2006) The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity. Toxicol In Vitro 20:785–792

    CAS  PubMed  Google Scholar 

  • Isom HC, Secott T, Georgoff I, Woodworth C, Mummaw J (1985) Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci USA 82:3252–3256

    CAS  PubMed  Google Scholar 

  • Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002) Mechanisms of hepatotoxicity. Toxicol Sci 65:166–176

    CAS  PubMed  Google Scholar 

  • Kasper HU, Dries V, Drebber U, Kern MA, Dienes HP, Schirmacher P (2005) Precision cut tissue slices of the liver as morphological tool for investigation of apoptosis. In Vivo 19:423–431

    PubMed  Google Scholar 

  • Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kim WH, Hong F, Jaruga B, Zhang ZS, Fan SJ, Liang TJ, Gao B (2005) Hepatitis B virus X protein sensitizes primary mouse hepatocytes to ethanol- and TNF-alpha-induced apoptosis by a caspase-3-dependent mechanism. Cell Mol Immunol 2:40–48

    CAS  PubMed  Google Scholar 

  • Kucera T, Canova NK, Farghali H, Martinek J (2006) The morphological and immunocytochemical evaluation of primary rat hepatocytes undergoing spontaneous cell death: modulation by the nitric oxide donor S-nitroso-N-acetylpenicillamine. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150:75–82

    CAS  PubMed  Google Scholar 

  • Kudo H, Takahara T, Yata Y, Kawai K, Zhang W, Sugiyama T (2009) Lipopolysaccharide triggered TNF-alpha-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model. J Hepatol 51:168–175

    CAS  PubMed  Google Scholar 

  • Kume E, Aruga C, Takahashi K, Miwa S, Dekura E, Itoh M, Ishizuka Y, Fujimura H, Toriumi W, Doi K (2005) Morphological and gene expression analysis in mouse primary cultured hepatocytes exposed to streptozotocin. Exp Toxicol Pathol 56:245–253

    CAS  PubMed  Google Scholar 

  • Lan L, Gorke S, Rau SJ, Zeisel MB, Hildt E, Himmelsbach K, Carvajal-Yepes M, Huber R, Wakita T, Schmitt-Graeff A, Royer C, Blum HE, Fischer R, Baumert TF (2008) Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner. J Immunol 181:4926–4935

    CAS  PubMed  Google Scholar 

  • Laurens M, Defamie V, Scozzari G, Schmid-Alliana A, Gugenheim J, Crenesse D (2005) Hypoxia-reoxygenation-induced chemokine transcription is not prevented by preconditioning or intermittent hypoxia, in mice hepatocytes. Transpl Int 18:444–452

    CAS  PubMed  Google Scholar 

  • Legembre P, Beneteau M, Daburon S, Moreau JF, Taupin JL (2003) Cutting edge: SDS-stable Fas microaggregates: an early event of Fas activation occurring with agonistic anti-Fas antibody but not with Fas ligand. J Immunol 171:5659–5662

    CAS  PubMed  Google Scholar 

  • Lei WW, Zhang KH, Pan XC, Wang DM, Hu Y, Yang YN, Song JG (2010) Histone deacetylase 1 and 2 differentially regulate apoptosis by opposing effects on extracellular signal-regulated kinase 1/2. Cell Death Dis 1:e44

    PubMed Central  PubMed  Google Scholar 

  • Luebke-Wheeler JL, Nedredal G, Yee L, Amiot BP, Nyberg SL (2009) E-cadherin protects primary hepatocyte spheroids from cell death by a caspase-independent mechanism. Cell Transplant 18:1281–1287

    PubMed Central  PubMed  Google Scholar 

  • Maeda S, Kimura H, Koga N, Lin KH, Saito T (1993) Cell density-dependent DNA fragmentation and its suppression by heparin in primary culture of adult rat hepatocytes. Biochem Biophys Res Commun 195:270–275

    CAS  PubMed  Google Scholar 

  • Malhi H, Barreyro FJ, Isomoto H, Bronk SF, Gores GJ (2007) Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity. Gut 56:1124–1131

    CAS  PubMed  Google Scholar 

  • Malhi H, Gores GJ (2008) Cellular and molecular mechanisms of liver injury. Gastroenterology 134:1641–1654

    CAS  PubMed Central  PubMed  Google Scholar 

  • Malhi H, Gores GJ, Lemasters JJ (2006) Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43:S31–S44

    CAS  PubMed  Google Scholar 

  • Martin H, Richert L, Berthelot A (2003) Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes. J Nutr 133:2505–2511

    CAS  PubMed  Google Scholar 

  • Meurette O, Fontaine A, Rebillard A, Le Moigne G, Lamy T, Lagadic-Gossmann D, Dimanche-Boitrel MT (2006) Cytotoxicity of TRAIL/anticancer drug combinations in human normal cells. Ann N Y Acad Sci 1090:209–216

    CAS  PubMed  Google Scholar 

  • Meurette O, Lefeuvre-Orfila L, Rebillard A, Lagadic-Gossmann D, Dimanche-Boitrel MT (2005) Role of intracellular glutathione in cell sensitivity to the apoptosis induced by tumor necrosis factor {alpha}-related apoptosis-inducing ligand/anticancer drug combinations. Clin Cancer Res 11:3075–3083

    CAS  PubMed  Google Scholar 

  • Michalopoulos GK, DeFrances M (2005) Liver regeneration. Adv Biochem Eng Biotechnol 93:101–134

    CAS  PubMed  Google Scholar 

  • Monte MJ, Marin JJ, Antelo A, Vazquez-Tato J (2009) Bile acids: chemistry, physiology, and pathophysiology. World J Gastroenterol 15:804–816

    CAS  PubMed  Google Scholar 

  • Musallam L, Ethier C, Haddad PS, Bilodeau M (2001) Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol 280:G1360–G1369

    CAS  PubMed  Google Scholar 

  • Muschen M, Warskulat U, Douillard P, Gilbert E, Haussinger D (1998) Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells. Hepatology 27:200–208

    CAS  PubMed  Google Scholar 

  • Nagata S (1999) Fas ligand-induced apoptosis. Annu Rev Genet 33:29–55

    CAS  PubMed  Google Scholar 

  • Nguyen LN, Furuya MH, Wolfraim LA, Nguyen AP, Holdren MS, Campbell JS, Knight B, Yeoh GC, Fausto N, Parks WT (2007) Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation. Hepatology 45:31–41

    CAS  PubMed  Google Scholar 

  • Ni R, Tomita Y, Matsuda K, Ichihara A, Ishimura K, Ogasawara J, Nagata S (1994) Fas-mediated apoptosis in primary cultured mouse hepatocytes. Exp Cell Res 215:332–337

    CAS  PubMed  Google Scholar 

  • Nipic D, Pirc A, Banic B, Suput D, Milisav I (2010) Preapoptotic cell stress response of primary hepatocytes. Hepatology 51:2140–2151

    CAS  PubMed  Google Scholar 

  • Oberhammer F, Froschl G, Tiefenbacher R, Inayat-Hussain SH, Cain K, Stopper H (1996) Hepatocyte death following transforming growth factor-beta 1 addition. Microsc Res Tech 34:247–258

    CAS  PubMed  Google Scholar 

  • Oberhammer FA, Pavelka M, Sharma S, Tiefenbacher R, Purchio AF, Bursch W, Schulte-Hermann R (1992) Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1. Proc Natl Acad Sci USA 89:5408–5412

    CAS  PubMed  Google Scholar 

  • Orrenius S, Nicotera P, Zhivotovsky B (2011) Cell death mechanisms and their implications in toxicology. Toxicol Sci 119:3–19

    CAS  PubMed  Google Scholar 

  • Ozaki M, Haga S, Zhang HQ, Irani K, Suzuki S (2003) Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1. Cell Death Differ 10:508–515

    CAS  PubMed  Google Scholar 

  • Pagliassotti MJ, Wei Y, Wang D (2007) Insulin protects liver cells from saturated fatty acid-induced apoptosis via inhibition of c-Jun NH2 terminal kinase activity. Endocrinology 148:3338–3345

    CAS  PubMed  Google Scholar 

  • Paine AJ, Andreakos E (2004) Activation of signalling pathways during hepatocyte isolation: relevance to toxicology in vitro. Toxicol In Vitro 18:187–193

    CAS  PubMed  Google Scholar 

  • Papeleu P, Vanhaecke T, Henkens T, Elaut G, Vinken M, Snykers S, Rogiers V (2006) Isolation of rat hepatocytes. Methods Mol Biol 320:229–237

    PubMed  Google Scholar 

  • Papeleu P, Wullaert A, Elaut G, Henkens T, Vinken M, Laus G, Tourwe D, Beyaert R, Rogiers V, Vanhaecke T (2007) Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2 N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes. Cell Prolif 40:640–655

    CAS  PubMed  Google Scholar 

  • Patel T, Bronk SF, Gores GJ (1994) Increases of intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. J Clin Invest 94:2183–2192

    CAS  PubMed Central  PubMed  Google Scholar 

  • Perez MJ, Briz O (2009) Bile-acid-induced cell injury and protection. World J Gastroenterol 15:1677–1689

    CAS  PubMed  Google Scholar 

  • Prince JM, Vodovotz Y, Baun MJ, Monga SP, Billiar TR, Gerlach JC (2010) The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support. Tissue Eng Part A 16:861–866

    CAS  PubMed  Google Scholar 

  • Prost S, Bellamy CO, Cunningham DS, Harrison DJ (1998) Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes. Faseb J 12:181–188

    CAS  PubMed  Google Scholar 

  • Pusl T, Wild N, Vennegeerts T, Wimmer R, Goke B, Brand S, Rust C (2008) Free fatty acids sensitize hepatocytes to bile acid-induced apoptosis. Biochem Biophys Res Commun 371:441–445

    CAS  PubMed  Google Scholar 

  • Qiao L, Farrell GC (1999) The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture. In Vitro Cell Dev Biol Anim 35:417–424

    CAS  PubMed  Google Scholar 

  • Raffray M, Cohen GM (1997) Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol Ther 75:153–177

    CAS  PubMed  Google Scholar 

  • Rautou PE, Mansouri A, Lebrec D, Durand F, Valla D, Moreau R (2010) Autophagy in liver diseases. J Hepatol 53:1123–1134

    CAS  PubMed  Google Scholar 

  • Realdon S, Gerotto M, Dal Pero F, Marin O, Granato A, Basso G, Muraca M, Alberti A (2004) Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation. J Hepatol 40:77–85

    CAS  PubMed  Google Scholar 

  • Reinehr R, Graf D, Fischer R, Schliess F, Haussinger D (2002) Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis. Hepatology 36:602–614

    CAS  PubMed  Google Scholar 

  • Ribeiro DH, Ferreira FL, da Silva VN, Aquino S, Correa B (2010) Effects of aflatoxin b(1) and fumonisin b(1) on the viability and induction of apoptosis in rat primary hepatocytes. Int J Mol Sci 11:1944–1955

    CAS  PubMed Central  PubMed  Google Scholar 

  • Rodriguez-Ariza A, Lopez-Sanchez LM, Gonzalez R, Corrales FJ, Lopez P, Bernardos A, Muntane J (2005) Altered protein expression and protein nitration pattern during d-galactosamine-induced cell death in human hepatocytes: a proteomic analysis. Liver Int 25:1259–1269

    CAS  PubMed  Google Scholar 

  • Rouquet N, Carlier K, Briand P, Wiels J, Joulin V (1996) Multiple pathways of Fas-induced apoptosis in primary culture of hepatocytes. Biochem Biophys Res Commun 229:27–35

    CAS  PubMed  Google Scholar 

  • Rowe C, Goldring CE, Kitteringham NR, Jenkins RE, Lane BS, Sanderson C, Elliott V, Platt V, Metcalfe P, Park BK (2010) Network analysis of primary hepatocyte dedifferentiation using a shotgun proteomics approach. J Proteome Res 9:2658–2668

    CAS  PubMed  Google Scholar 

  • Sanchez A, Alvarez AM, Benito M, Fabregat I (1996) Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive oxygen intermediates. J Biol Chem 271:7416–7422

    CAS  PubMed  Google Scholar 

  • Schattenberg JM, Galle PR, Schuchmann M (2006) Apoptosis in liver disease. Liver Int 26:904–911

    CAS  PubMed  Google Scholar 

  • Schlatter R, Schmich K, Lutz A, Trefzger J, Sawodny O, Ederer M, Merfort I (2011) Modeling the TNFalpha-induced apoptosis pathway in hepatocytes. PLoS ONE 6:e18646

    CAS  PubMed Central  PubMed  Google Scholar 

  • Schlosser SF, Azzaroli F, Dao T, Hingorani R, Nicholas Crispe I, Boyer JL (2000) Induction of murine hepatocyte death by membrane-bound CD95 (Fas/APO-1)-ligand: characterization of an in vitro system. Hepatology 32:779–785

    CAS  PubMed  Google Scholar 

  • Schmich K, Schlatter R, Corazza N, Sa Ferreira K, Ederer M, Brunner T, Borner C, Merfort I (2011) Tumor necrosis factor alpha sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner. Hepatology 53:282–292

    CAS  PubMed  Google Scholar 

  • Schrenk D, Schmitz HJ, Bohnenberger S, Wagner B, Worner W (2004) Tumor promoters as inhibitors of apoptosis in rat hepatocytes. Toxicol Lett 149:43–50

    CAS  PubMed  Google Scholar 

  • Schulze-Bergkamen H, Brenner D, Krueger A, Suess D, Fas SC, Frey CR, Dax A, Zink D, Buchler P, Muller M, Krammer PH (2004) Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt. Hepatology 39:645–654

    CAS  PubMed  Google Scholar 

  • Schulze-Bergkamen H, Schuchmann M, Fleischer B, Galle PR (2006) The role of apoptosis versus oncotic necrosis in liver injury: facts or faith? J Hepatol 44:984–993

    CAS  PubMed  Google Scholar 

  • Schulze-Bergkamen H, Untergasser A, Dax A, Vogel H, Buchler P, Klar E, Lehnert T, Friess H, Buchler MW, Kirschfink M, Stremmel W, Krammer PH, Muller M, Protzer U (2003) Primary human hepatocytes—a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol 38:736–744

    PubMed  Google Scholar 

  • Schwabe RF, Uchinami H, Qian T, Bennett BL, Lemasters JJ, Brenner DA (2004) Differential requirement for c-Jun NH2-terminal kinase in TNFalpha- and Fas-mediated apoptosis in hepatocytes. Faseb J 18:720–722

    CAS  PubMed  Google Scholar 

  • Schwall RH, Robbins K, Jardieu P, Chang L, Lai C, Terrell TG (1993) Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology 18:347–356

    CAS  PubMed  Google Scholar 

  • Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83

    CAS  PubMed  Google Scholar 

  • Shimizu S, Eguchi Y, Kamiike W, Akao Y, Kosaka H, Hasegawa J, Matsuda H, Tsujimoto Y (1996) Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes. Am J Physiol 271:G949–G958

    CAS  PubMed  Google Scholar 

  • Shinzawa K, Watanabe Y, Akaike T (1995) Primary cultured murine hepatocytes but not hepatoma cells regulate the cell number through density-dependent cell death. Cell Death Differ 2:133–140

    CAS  PubMed  Google Scholar 

  • Smets FN, Chen Y, Wang LJ, Soriano HE (2002) Loss of cell anchorage triggers apoptosis (anoikis) in primary mouse hepatocytes. Mol Genet Metab 75:344–352

    CAS  PubMed  Google Scholar 

  • St-Pierre MV, Dufour JF (2012) Biomarkers for hepatocellular apoptosis in the management of liver diseases. Curr Pharm Biotechnol 13:2221–2227

    PubMed  Google Scholar 

  • Terradillos O, Pollicino T, Lecoeur H, Tripodi M, Gougeon ML, Tiollais P, Buendia MA (1998) p53-independent apoptotic effects of the hepatitis B virus HBx protein in vivo and in vitro. Oncogene 17:2115–2123

    CAS  PubMed  Google Scholar 

  • Thilenius AR, Braun K, Russell JH (1997) Agonist antibody and Fas ligand mediate different sensitivity to death in the signaling pathways of Fas and cytoplasmic mutants. Eur J Immunol 27:1108–1114

    CAS  PubMed  Google Scholar 

  • Tichy M, Pokorna A, Hanzlikova I, Nerudova J, Tumova J, Uzlova R (2010) Primary rat hepatocytes in chemical testing and QSAR predictive applicability. Toxicol In Vitro 24:240–244

    CAS  PubMed  Google Scholar 

  • 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 181:124–137

    CAS  PubMed  Google Scholar 

  • Vanhaecke T, Henkens T, Kass GE, 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

    CAS  PubMed  Google Scholar 

  • Vanhaecke T, Henkens T, Snykers S, Elaut G, Papeleu P, Rogiers V (2006) Effects of Trichostatin A on apoptosis-regulating proteins during hepatocyte isolation. ALTEX 23:441–444

    Google Scholar 

  • Vinken M, Papeleu P, Snykers S, De Rop E, Henkens T, Chipman JK, Rogiers V, Vanhaecke T (2006) Involvement of cell junctions in hepatocyte culture functionality. Crit Rev Toxicol 36:299–318

    CAS  PubMed  Google Scholar 

  • Vinken M, Decrock E, De Vuyst E, Leybaert L, Vanhaecke T, Rogiers V (2009) Biochemical characterisation of an in vitro model of hepatocellular apoptotic cell death. Altern Lab Anim 37:209–218

    CAS  PubMed  Google Scholar 

  • Vinken M, Snykers S, Fraczek J, Decrock E, Leybaert L, Rogiers V, Vanhaecke T (2010) DNA methyltransferase 3a expression decreases during apoptosis in primary cultures of hepatocytes. Toxicol In Vitro 24:445–451

    CAS  PubMed  Google Scholar 

  • Vinken M, Decrock E, Doktorova T, Ramboer E, De Vuyst E, Vanhaecke T, Leybaert L, Rogiers V (2011) Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes. Arch Toxicol 85:1589–1596

    CAS  PubMed  Google Scholar 

  • Vinken M, Decrock E, Vanhaecke T, Leybaert L, Rogiers V (2012a) Connexin43 signaling contributes to spontaneous apoptosis in cultures of primary hepatocytes. Toxicol Sci 125:175–186

    CAS  PubMed  Google Scholar 

  • Vinken M, Vanhaecke T, Rogiers V (2012b) Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? Toxicol In Vitro 26:541–544

    CAS  PubMed  Google Scholar 

  • Walter D, Schmich K, Vogel S, Pick R, Kaufmann T, Hochmuth FC, Haber A, Neubert K, McNelly S, von Weizsacker F, Merfort I, Maurer U, Strasser A, Borner C (2008) Switch from type II to I Fas/CD95 death signaling on in vitro culturing of primary hepatocytes. Hepatology 48:1942–1953

    CAS  PubMed Central  PubMed  Google Scholar 

  • Wang JM, Wang H, Xu LN, Zou SQ (2005) Hepatic injury in rats with obstructive jaundice: roles of the protein kinase C signal pathway and cytoprotection of fructose. Hepatobiliary Pancreat Dis Int 4:577–581

    CAS  PubMed  Google Scholar 

  • Wang K, Brems JJ, Gamelli RL, Holterman AX (2011) iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes. Cell Signal 23:1677–1685

    CAS  PubMed  Google Scholar 

  • Wang K, Brems JJ, Gamelli RL, Holterman AX (2010) Survivin signaling is regulated through nuclear factor-kappa B pathway during glycochenodeoxycholate-induced hepatocyte apoptosis. Biochim Biophys Acta 1803:1368–1375

    CAS  PubMed  Google Scholar 

  • Worner W, Schrenk D (1996) Influence of liver tumor promoters on apoptosis in rat hepatocytes induced by 2-acetylaminofluorene, ultraviolet light, or transforming growth factor beta 1. Cancer Res 56:1272–1278

    CAS  PubMed  Google Scholar 

  • Yang D, Liu N, Zuo C, Lei S, Wu X, Zhou F, Liu C, Zhu H (2011) Innate host response in primary human hepatocytes with hepatitis C virus infection. PLoS ONE 6:e27552

    CAS  PubMed Central  PubMed  Google Scholar 

  • Yao Y, Zhang D, Luo Y, Zhang D, Huang A, Zhou W, Ren H (2000) Fas ligand expression and apoptosis in primary rat hepatocytes induced by lipopolysaccharide. Zhonghua Gan Zang Bing Za Zhi 8:285–287

    CAS  PubMed  Google Scholar 

  • Yoo J, Ghiassi M, Jirmanova L, Balliet AG, Hoffman B, Fornace AJ Jr, Liebermann DA, Bottinger EP, Roberts AB (2003) Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J Biol Chem 278:43001–43007

    CAS  PubMed  Google Scholar 

  • Zucchini N, de Sousa G, Bailly-Maitre B, Gugenheim J, Bars R, Lemaire G, Rahmani R (2005) Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes. Biochim Biophys Acta 1745:48–58

    CAS  PubMed  Google Scholar 

  • Zucchini-Pascal N, de Sousa G, Pizzol J, Rahmani R (2011) Molecular investigation of the effects of lindane in rat hepatocytes: microarray and mechanistic studies. Food Chem Toxicol 49:3128–3135

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the grants of the University Hospital of the Vrije Universiteit Brussel (Willy Gepts Fonds UZ-VUB), the Fund for Scientific Research Flanders (FWO-Vlaanderen), the European Union (FP7/Cosmetics Europe projects HeMiBio and DETECTIVE) and the European Research Council (CONNECT project).

Author information

Authors and Affiliations

  1. Department of Toxicology (FAFY), Faculty of Medicine and Pharmacy, Center for Pharmaceutical Research (CePhaR), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium

    Mathieu Vinken, Michaël Maes, Tamara Vanhaecke & Vera Rogiers

  2. Department of Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil

    André G. Oliveira, Pedro E. Marques & Gustavo B. Menezes

  3. Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil

    Bruno Cogliati & Maria Lúcia Zaidan Dagli

Authors
  1. Mathieu Vinken
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michaël Maes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André G. Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruno Cogliati
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pedro E. Marques
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gustavo B. Menezes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maria Lúcia Zaidan Dagli
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tamara Vanhaecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Vera Rogiers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mathieu Vinken.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vinken, M., Maes, M., Oliveira, A.G. et al. Primary hepatocytes and their cultures in liver apoptosis research. Arch Toxicol 88, 199–212 (2014). https://doi.org/10.1007/s00204-013-1123-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-013-1123-4

Keywords

Search

Navigation