Precision-Cut Liver Slices as anex vivomodel to evaluate antifibrotic therapies for liver fibrosis and cirrhosis

Abstract

AbstractBackgroundPrecision-Cut Liver Slices (PCLS) are anex vivoculture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximatesin vivopathology thanin vitromethods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS.MethodsPCLS of 250 μm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 μM Erlotinib for 72 hours. Histology and gene expression were then compared within vivomurine experiments and TGF-β1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver.ResultsPCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genesIl6,Col1a1andTimp1in PCLS from CDAHFD-induced cirrhotic mice, andIl6,Col1a1andTgfb1in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression ofCol1a1,Timp1,Tgfb1andIl6, which was consistent with the impact of erlotinib onCol1a1andTgfb1expression inin vivoDEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression ofTimp1,Col1a1andTgfb1, which was consistent with the effect of erlotinib inin vivoCCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-β1 treatment upregulatedActa2(αSMA), while treatment with erlotinib inhibited the expression ofActa2. Similar expression results were observed in TGF-β1 treatedin vitroHSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples.ConclusionThe responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observedin vivoandin vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).