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Arch Toxicol. 2015 Oct;89(10):1861-70. doi: 10.1007/s00204-015-1575-9. Epub 2015 Aug 18.

Bile canalicular dynamics in hepatocyte sandwich cultures.

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Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU Dortmund, Dortmund, Germany.
Fraunhofer-Chalmers Centre, Chalmers Science Park, Göteborg, Sweden.
Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU Dortmund, Dortmund, Germany.
Centre for Immunology and Infection, University of York, York, UK.
Immunology and Infection Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.
Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.
Department of Pharmacy and Biotechnology, Molecular Toxicology Unit, University of Bologna, Bologna, Italy.
Fraunhofer-Chalmers Centre, Chalmers Science Park, Göteborg, Sweden.


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.


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

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