Organotypic 3D HepaRG Liver Model for Assessment of Drug-Induced Cholestasis

: Cholestasis remains a major challenge in drug-induced liver injury, and therefore warrants identification of chemical entities that may lead to cholestasis. Recent advances in cell culture methods enable 3D spheroid models to remain viable for much longer periods of time than conventional sandwich cultures of primary human hepatocytes while maintaining native tissue-like functionality, such as drug metabolism activity, receptor signaling functionality, and physiological relevance. These spheroid models enable us to study repeated exposure effects associated with chemicals and their metabolites that may ultimately progress to cholestasis and liver injury. HepaRG cells cultured as spheroids are viable for more than 4 weeks with cytochrome P450 enzymatic activities comparable to ranges observed in freshly isolated/cryopreserved suspensions of primary human hepatocytes. HepaRG spheroids form bile canalicular structures with potential application as a model to study biliary excretion processes and intrahepatic obstruction of bile flow, leading to hepatocellular damage and death. In this chapter, we describe methods to culture 3D spheroids of HepaRG cells with extensive bile canalicular structures/networks, image transport of bile acid (cholyl-lysyl-fluorescein) to the bile canaliculi, and measure cholestatic drug-induced cytotoxicity.