Hepatotoxic Response in 2D and 3D Co-culture Models Differs From Hepatocyte-alone Models

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Authors: Allysa Stern, Karissa Marino, Tyler Beames, Amy Stefanowicz, Michael Black, Patrick McMullen, Scott Slattery, and Jessica Hartman

Safety assessment strategies that rely on testing species-relevant cells in vitro are advancing, yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators. An organotypic rat model, capable of predicting rat in vivo responses to hepatotoxicants, will provide an affordable and ethical alternative to in vivo rat models currently utilized. An ideal in vitro liver model for hepatotoxicity testing would include hepatocytes and the non-parenchymal cells (NPCs) (i.e., hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells), and would support hepatocyte viability, phenotypic maintenance, and metabolic competence for an extended time in culture to allow for repeated exposures and long-term dosing. The current studies have focused on developing organotypic two-dimensional 96-well plate-based and three-dimensional alginate bead-based culture systems that include primary rat hepatocytes and NPCs and can support hepatocyte viability in vitro out to eight days in 2D and beyond 21 days in 3D. Using several canonical hepatotoxicants, we have compared 2D and 3D hepatocyte-alone (mono-) and hepatocyte and NPC (co-) culture systems to determine the robustness of these models. After treatment with acetaminophen (APAP; 1 mM, 5 mM, 10 mM, 15 mM 20 mM) for 3 consecutive days, APAP-induced cytotoxicity was significantly increased in the mono-culture model, compared to the co-culture model. Transcriptomic analysis of 2D mono- and co-culture models revealed that co-culture response to phenobarbital (10 ┬ÁM) is more representative (32% increase in similarity of the ontology enrichment) of in vivo responses (using OPEN TG_GATEs in vivo data, 300 mg/kg, 24 h). These studies have demonstrated that the organotypic model can recapitulate rodent in vivo liver phenotypes observed in response to canonical hepatotoxicants and suggest that the co-culture model could be useful for testing the effects of compounds in vitro as an early stage alternative to in-life studies.