Developing an in vitro screening assay platform for evaluation of antifibrotic drugs using precision-cut liver slices

Background: Precision-cut liver slices present different cell types of liver in a physiological context, and they have been explored as effective in vitro model systems to study liver fibrosis. Inducing fibrosis in the liver slices using toxicants like carbon tetrachloride is of less relevance to human disease conditions. Our aim for this study was to establish physiologically relevant conditions in vitro to induce fibrotic phenotypes in the liver slices. Results: Precision-cut liver slices of 150 mu m thickness were obtained from female C57BL/6 J mice. The slices were cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-beta, PDGF, 5 mu M each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 mu g/ml of lipopolysaccharide along with 500 mu M of palmitate and were analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes )IL-6, TNF-alpha and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin. Conclusions: This study reports the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system presented here captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen small molecules for their antifibrotic activity.