Development of 2D organotypic rat liver co-culture models for assessing hepatotoxicant modes of action

In both the regulatory and commercial arenas, the goal is to move away from time consuming, costly in-life rodent studies and towards safety assessment strategies that rely on testing species-relevant cells in vitro. The liver has been a major focus of these efforts, yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators, and the assays that do exist typically utilize hepatocyte monolayer culture. While hepatocytes have been the primary component of in vitro hepatotoxicity assay development, the non-parenchymal cells (NPCs) (i.e., hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells) also play a critical role in the progression of liver pathologies. The goal of this study was to develop an organotypic co-culture system that could read out the various mechanisms of action observed in vivo, as well as maintain metabolic capability and extended viability to allow for repeat dosing. We developed a two-dimensional 96-well plate-based co-culture system that includes primary rat hepatocytes, stellate, Kupffer, and endothelial cells that supports hepatocyte viability and phenotype stability for up to eight days. Importantly, markers of hepatocyte differentiation and polarity are maintained in co-culture, but hepatocyte monocultures with no other cell types lose phenotypic markers after prolonged culture. This co-culture model was leveraged to assess the effects of known hepatotoxic stimuli, and the co-culture model resulted in responses that were more representative of the in vivo phenotypes. Overall design: Freshly isolated male rat Sprague Dawley hepatocytes and non-parenchymal cells were obtained from Lonza (Walkersville, MD) as described. The cells were cultured in either 2D mono- or co-culture and treated with either Ketoconazole (1, 10, 30 µM), GW7647 (0.01, 0.1, 1 µM), Gemfibrozil (10, 100, 1000 µM), or Phenobarbital (0.1, 1, 10 mM). The cells were treated 24 hours prior to sample collection at 3 timepoints (days 1, 3, and 7 post plating). After 24 hours, the cells were lysed in TempO-Seq buffer and libraries were prepared for sequencing per manufacturer's protocols (n=4 biological replicates).