Abstract
BackgroundPrecision-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.ResultsPrecision-cut liver slices of 150 μm 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-β, PDGF, 5 μM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 μg/ml of lipopolysaccharide along with 500 μM 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-α and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin.ConclusionsThis 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.
Highlights
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
We report on developing liver fibrosis in liver slices using physiological signals that will activate key signaling pathways effectively and result in important end points relevant to NAFLD/fibrosis triglyceride accumulation, hepatocyte dysfunction and inflammation, hepatic stellate cell activation, and extracellular matrix (ECM) remodeling with increased collagen production
Several signaling pathways are activated during pathogenesis of fibrosis, and each of these pathways contributes at various stages of the pathology leading to hepatic stellate cell activation and ECM production
Summary
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. Several etiological factors like viral infection, alcohol abuse, insulin resistance and metabolic disorder contribute to the development of fibrotic phenotype [1]. It is a complex process involving various cell types of liver including hepatocytes, several immune cell types and stellate cells [2,3]. Cell lines and isolated primary cultures serve as good model systems to address mechanism-based questions and to understand the cell type-specific biology They fail to represent the liver as a multicellular system in which several cell types and cell-cell interactions contribute toward fibrogenesis [5].
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