A 3D in vitro screening-based discovery approach for selecting and prioritizing NASH drug candidates Simon Ströbel, Radina Kostadinova, Katia Fiaschetti, Jana Rupp, Agnieszka Pajak, Katarzyna Sanchez, Armin Wolf, Eva Thoma InSphero AG, Schlieren, Switzerland Non-alcoholic steatohepatitis is a severe progressive disease characterized by fat accumulation, inflammation, and fibrosis in the liver. To date, there are no approved drugs for NASH treatment and drug development has been impeded by the lack of predictive in vitro models reflecting the complex pathology of NASH. The aim of the study was to develop a human 3D in vitro NASH discovery platform, suitable for screening and prioritization of NASH drug candidates. We engineered a scalable model system (appropriate for screening assays in 96- and 384-well plates), based on a scaffold-free co-culture of the liver cell types needed to recapitulate the NASH disease progression in patients: primary hepatocytes, Kupffer cells, liver endothelial cells, and hepatic stellate cells. Using this 3D in vitro model system, we induced NASH using a cocktail of lipotoxic and inflammatory stimuli (free fatty acids and LPS) in media containing high levels of sugar and insulin. Compared to untreated controls, disease-induced models displayed key pathophysiological features of NASH after 10 days of treatment: 1) increase of intracellular triglyceride content as an indicator of fat accumulation; 2) secretion of inflammatory cytokines/chemokines/growth factors, such as IL-6, TNF-α, MIP-1α, IP-10, MCP-1, IL-8; VEGF, and 3) increased fibril collagen deposition and secretion of procollagen type I and III peptides. Whole transcriptome analysis of NASH-treated models versus control revealed activation of pathways and differential regulation of genes associated with key processes in NASH, including lipid metabolism, inflammation, and fibrosis. Treatment with anti-TGF-β antibody and ALK5i (TGFβRI inhibitor) decreased secretion of procollagen type I/III in a concentration dependent manner. Decreased deposition of fibril collagens, as quantified by Sirius-Red staining (PharmaNest), was observed in models treated with anti-TGF-β antibody and ALK5i. Importantly, biochemical readouts for 3D models treated with NASH drug candidates (Selonsertib and Firsocostat) were indicative of disease progression, and our results generally reflected documented clinical observations. In summary, this human 3D in vitro discovery platform is a promising research tool for selection of most effective novel anti-NASH drug candidates to advance in the NASH development pipeline.