Development and validation of an in vitro 3D model of NASH with severe fibrotic phenotype.

Abstract

Nonalcoholic steatohepatitis represents a significant and rapidly growing unmet medical need. The development of novel therapies has been hindered in part, by the limitations of existing preclinical models. There is a strong need for physiologically relevant <i>in vivo</i> and <i>in vitro</i> liver fibrosis models that are characterized by better translational predictability. In this study, we used the InSphero 3D InSight^TM three-dimensional (3D) human liver microtissue (3D-hLMT) system prepared by co-culturing primary human hepatocytes with hepatic stellate cells, Kupffer cells and endothelial cells to develop a model of NASH with a severe fibrotic phenotype. In our model, palmitic acid (PA) induced a robust proinflammatory and profibrogenic phenotype in the 3D-hLMT. PA significantly increased several markers of the inflammatory and profibrotic process including gene expression of collagens, <i>α-sma</i>, tissue inhibitor of matrix metalloprotease 1 (<i>timp1</i>) and the stellate cell activation marker <i>pdgfrβ</i> as well as secreted CXCL8 (IL8) levels. We also observed TGFβ pathway activation, increase in active collagen synthesis and significant overall increase in tissue damage in the 3D-hLMTs. Immunohistochemistry analysis demonstrated the upregulation of collagen, cleaved caspase 3 as well as of the PDGFRβ protein. We further validated the model using a phase 3 clinical compound, GS-4997, an apoptosis signal-regulating kinase 1 (ASK-1) inhibitor and showed that GS-4997 significantly decreased PA induced profibrotic and proinflammatory response in the 3D-hLMTs with decreases in apoptosis and stellate cell activation in the microtissues. Taken together we have established and validated an <i>in vitro</i> 3D-hLMT NASH model with severe fibrotic phenotype that can be a powerful tool to investigate experimental compounds for the treatment of NASH.