Archives of Toxicology

, Volume 89, Issue 10, pp 1861–1870 | Cite as

Bile canalicular dynamics in hepatocyte sandwich cultures

  • Raymond Reif
  • Johan Karlsson
  • Georgia Günther
  • Lynette Beattie
  • David Wrangborg
  • Seddik Hammad
  • Brigitte Begher-Tibbe
  • Amruta Vartak
  • Simone Melega
  • Paul M. Kaye
  • Jan G. Hengstler
  • Mats Jirstrand
In vitro systems

Abstract

Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.

Keywords

Collagen sandwich Bile canaliculi Cholestasis Primary hepatocytes Image quantification 3D system 

Notes

Funding

This work was partially funded by the European Commission Seventh Framework Programme CANCERSYS (FP7-2008-2011; Grant #223188), NOTOX (FP7-2007-2013; Grant #267038), DETECTIVE (FP7-2007-2013; Grant #266838), the Virtual Liver Network initiative of the German Federal Ministry of Education and Research (Grant #03157399) and by the UK Medical Research Council (Grant #G0802620).

Compliance with ethical standards

Conflict of interest

None.

Supplementary material

204_2015_1575_MOESM1_ESM.xlsx (334 kb)
Area.xlsx Determined bile canalicular area for the control and dex/ins cultures (XLSX 333 kb)
204_2015_1575_MOESM2_ESM.xlsx (23 kb)
Morphology.xlsx Quantified morphology of hepatocytes in culture and in vivo (XLSX 22 kb)
204_2015_1575_MOESM3_ESM.mpg (3.5 mb)
Video1.mpg Maturation process of a control sandwich culture (MPG 3614 kb)
204_2015_1575_MOESM4_ESM.mpg (3.3 mb)
Video2.mpg CMFDA excretion into bile canaliculi in vivo (MPG 3368 kb)
204_2015_1575_MOESM5_ESM.mpg (3.1 mb)
Video3.mpg CMFDA excretion into bile canaliculi sandwich culture (MPG 3130 kb)
204_2015_1575_MOESM6_ESM.mpg (7.7 mb)
Video4.mpg Segmentation and quantification of bile canalicular volume (MPG 7862 kb)
Video5.mpg

Hepatocyte sandwich culture control 1 (MPG 1608 kb)

204_2015_1575_MOESM8_ESM.mpg (5.5 mb)
Video6.mpg Hepatocyte sandwich culture control 2 (MPG 5658 kb)
204_2015_1575_MOESM9_ESM.mpg (7.4 mb)
Video7.mpg Hepatocyte sandwich culture control 3 (MPG 7568 kb)
204_2015_1575_MOESM10_ESM.mpg (8.8 mb)
Video8.mpg Hepatocyte sandwich culture control 4 (MPG 9006 kb)
Video9.mpg

Hepatocyte sandwich supplemented with dexamethason insulin 1 (MPG 2414 kb)

204_2015_1575_MOESM12_ESM.mpg (11.6 mb)
Video10.mpg Hepatocyte sandwich supplemented with dexamethason insulin 2 (MPG 11904 kb)
204_2015_1575_MOESM13_ESM.mpg (7.1 mb)
Video11.mpg Hepatocyte sandwich supplemented with dexamethason insulin 3 (MPG 7254 kb)
204_2015_1575_MOESM14_ESM.mpg (6.7 mb)
Video12.mpg Hepatocyte sandwich supplemented with dexamethason insulin 4 (MPG 6868 kb)
204_2015_1575_MOESM15_ESM.mpg (9 mb)
Video13.mpg Hepatocyte sandwich supplemented with dexamethason insulin 5 (MPG 9212 kb)

References

  1. Beattie L, Peltan A, Maroof A et al (2010) Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells. PLoS Pathog 6(3):e1000805. doi: 10.1371/journal.ppat.1000805 PubMedCentralCrossRefPubMedGoogle Scholar
  2. Bi YA, Kazolias D, Duignan DB (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos 34(9):1658–1665. doi: 10.1124/dmd.105.009118 CrossRefPubMedGoogle Scholar
  3. 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(2):174–177PubMedGoogle Scholar
  4. Ekani-Nkodo A, Fygenson DK (2003) Size exclusion and diffusion of fluoresceinated probes within collagen fibrils. Phys Rev E 67(2 Pt 1):021909CrossRefGoogle Scholar
  5. Erlinger S (1978) Cholestasis: pump failure, microvilli defect, or both? Lancet 1(8063):533–534CrossRefPubMedGoogle Scholar
  6. Godoy P, Hengstler JG, Ilkavets I et al (2009) Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 49(6):2031–2043. doi: 10.1002/hep.22880 CrossRefPubMedGoogle Scholar
  7. Godoy P, Hewitt NJ, Albrecht U et al (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. doi: 10.1007/s00204-013-1078-5 PubMedCentralCrossRefPubMedGoogle Scholar
  8. Hammad S, Hoehme S, Friebel A et al (2014) Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis. Arch Toxicol 88(5):1161–1183. doi: 10.1007/s00204-014-1243-5 PubMedCentralCrossRefPubMedGoogle Scholar
  9. Hewitt NJ, Lechon MJ, Houston JB et al (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. doi: 10.1080/03602530601093489 CrossRefPubMedGoogle Scholar
  10. Kostrubsky VE, Strom SC, Hanson J et al (2003) Evaluation of hepatotoxic potential of drugs by inhibition of bile-acid transport in cultured primary human hepatocytes and intact rats. Toxicol Sci 76(1):220–228. doi: 10.1093/toxsci/kfg217 CrossRefPubMedGoogle Scholar
  11. Kotani N, Maeda K, Watanabe T et al (2011) Culture period-dependent changes in the uptake of transporter substrates in sandwich-cultured rat and human hepatocytes. Drug Metab Dispos 39(9):1503–1510. doi: 10.1124/dmd.111.038968 CrossRefPubMedGoogle Scholar
  12. LeCluyse EL, Audus KL, Hochman JH (1994) Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration. Am J Physiol 266(6 Pt 1):C1764–C1774PubMedGoogle Scholar
  13. Liu X, Chism JP, LeCluyse EL, Brouwer KR, Brouwer KL (1999a) Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab Dispos 27(6):637–644PubMedGoogle Scholar
  14. Liu X, LeCluyse EL, Brouwer KR et al (1999b) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 277(1 Pt 1):G12–G21PubMedGoogle Scholar
  15. Liu X, LeCluyse EL, Brouwer KR, Lightfoot RM, Lee JI, Brouwer KL (1999c) Use of Ca2+ modulation to evaluate biliary excretion in sandwich-cultured rat hepatocytes. J Pharmacol Exp Ther 289(3):1592–1599PubMedGoogle Scholar
  16. Luttringer O, Theil FP, Lave T, Wernli-Kuratli K, Guentert TW, de Saizieu A (2002) Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem Pharmacol 64(11):1637–1650CrossRefPubMedGoogle Scholar
  17. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605. doi: 10.1002/dvg.20335 CrossRefPubMedGoogle Scholar
  18. Oda M, Phillips MJ (1977) Bile canalicular membrane pathology in cytochalasin B-induced cholestasis. Lab Invest 37(4):350–356PubMedGoogle Scholar
  19. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3):313–319CrossRefPubMedGoogle Scholar
  20. Oshio C, Phillips MJ (1981) Contractility of bile canaliculi: implications for liver function. Science 212(4498):1041–1042CrossRefPubMedGoogle Scholar
  21. Padda MS, Sanchez M, Akhtar AJ, Boyer JL (2011) Drug-induced cholestasis. Hepatology 53(4):1377–1387. doi: 10.1002/hep.24229 PubMedCentralCrossRefPubMedGoogle Scholar
  22. Phillips MJ, Oshio C, Miyairi M, Katz H, Smith CR (1982) A study of bile canalicular contractions in isolated hepatocytes. Hepatology 2(6):763–768CrossRefPubMedGoogle Scholar
  23. Rippin SJ, Hagenbuch B, Meier PJ, Stieger B (2001) Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes. Hepatology 33(4):776–782. doi: 10.1053/jhep.2001.23433 CrossRefPubMedGoogle Scholar
  24. Thibault N, Claude JR, Ballet F (1992) Actin filament alteration as a potential marker for cholestasis: a study in isolated rat hepatocyte couplets. Toxicology 73(3):269–279CrossRefPubMedGoogle Scholar
  25. Watanabe N, Tsukada N, Smith CR, Phillips MJ (1991) Motility of bile canaliculi in the living animal: implications for bile flow. J Cell Biol 113(5):1069–1080CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Raymond Reif
    • 1
  • Johan Karlsson
    • 2
  • Georgia Günther
    • 1
  • Lynette Beattie
    • 3
    • 6
  • David Wrangborg
    • 2
  • Seddik Hammad
    • 1
    • 4
  • Brigitte Begher-Tibbe
    • 1
  • Amruta Vartak
    • 1
  • Simone Melega
    • 5
  • Paul M. Kaye
    • 3
  • Jan G. Hengstler
    • 1
  • Mats Jirstrand
    • 2
  1. 1.Leibniz Research Centre for Working Environment and Human Factors (IfADo)TU DortmundDortmundGermany
  2. 2.Fraunhofer-Chalmers CentreGöteborgSweden
  3. 3.Centre for Immunology and InfectionUniversity of YorkYorkUK
  4. 4.Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary MedicineSouth Valley UniversityQenaEgypt
  5. 5.Department of Pharmacy and Biotechnology, Molecular Toxicology UnitUniversity of BolognaBolognaItaly
  6. 6.Immunology and Infection LaboratoryQIMR Berghofer Medical Research InstituteHerstonAustralia

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