Bile canalicular dynamics in hepatocyte sandwich cultures
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.
KeywordsCollagen 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
Hepatocyte sandwich culture control 1 (MPG 1608 kb)
Hepatocyte sandwich supplemented with dexamethason insulin 1 (MPG 2414 kb)
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