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Bile canalicular dynamics in hepatocyte sandwich cultures

  • In vitro systems
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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.

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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).

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Author notes

  1. Lynette Beattie

    Present address: Immunology and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia

Authors and Affiliations

  1. Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU Dortmund, Dortmund, Germany

    Raymond Reif, Georgia Günther, Seddik Hammad, Brigitte Begher-Tibbe, Amruta Vartak & Jan G. Hengstler

  2. Fraunhofer-Chalmers Centre, Chalmers Science Park, Göteborg, Sweden

    Johan Karlsson, David Wrangborg & Mats Jirstrand

  3. Centre for Immunology and Infection, University of York, York, UK

    Lynette Beattie & Paul M. Kaye

  4. Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

    Seddik Hammad

  5. Department of Pharmacy and Biotechnology, Molecular Toxicology Unit, University of Bologna, Bologna, Italy

    Simone Melega

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  1. Raymond Reif
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  2. Johan Karlsson
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Corresponding authors

Correspondence to Raymond Reif or Mats Jirstrand.

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Additional information

Raymond Reif and Johan Karlsson have contributed equally to this work. Mats Jirstrand and Jan G. Hengstler have a shared senior authorship.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Area.xlsx

Determined bile canalicular area for the control and dex/ins cultures (XLSX 333 kb)

Morphology.xlsx

Quantified morphology of hepatocytes in culture and in vivo (XLSX 22 kb)

Video1.mpg

Maturation process of a control sandwich culture (MPG 3614 kb)

Video2.mpg

CMFDA excretion into bile canaliculi in vivo (MPG 3368 kb)

Video3.mpg

CMFDA excretion into bile canaliculi sandwich culture (MPG 3130 kb)

Video4.mpg

Segmentation and quantification of bile canalicular volume (MPG 7862 kb)

Hepatocyte sandwich culture control 1 (MPG 1608 kb)

Video6.mpg

Hepatocyte sandwich culture control 2 (MPG 5658 kb)

Video7.mpg

Hepatocyte sandwich culture control 3 (MPG 7568 kb)

Video8.mpg

Hepatocyte sandwich culture control 4 (MPG 9006 kb)

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

Video10.mpg

Hepatocyte sandwich supplemented with dexamethason insulin 2 (MPG 11904 kb)

Video11.mpg

Hepatocyte sandwich supplemented with dexamethason insulin 3 (MPG 7254 kb)

Video12.mpg

Hepatocyte sandwich supplemented with dexamethason insulin 4 (MPG 6868 kb)

Video13.mpg

Hepatocyte sandwich supplemented with dexamethason insulin 5 (MPG 9212 kb)

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Reif, R., Karlsson, J., Günther, G. et al. Bile canalicular dynamics in hepatocyte sandwich cultures. Arch Toxicol 89, 1861–1870 (2015). https://doi.org/10.1007/s00204-015-1575-9

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  • DOI: https://doi.org/10.1007/s00204-015-1575-9

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