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


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.


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



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


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)

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)

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)


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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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