bioRxiv2018
Summary boxO_LIWhat is already known about this subject?\nO_LICurrently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.\nC_LIO_LIPreclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.\nC_LIO_LIPrecision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.\nC_LI\nC_LIO_LIWhat are the new findings?\nO_LIWe have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.\nC_LIO_LIBioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.\nC_LIO_LIAdministration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.\nC_LI\nC_LIO_LIHow might it impact on clinical practice in the foreseeable future?\nO_LIThe extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.\nC_LIO_LIBioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.\nC_LI\nC_LI\n\nAbstractO_ST_ABSObjectiveC_ST_ABSPrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.\n\nDesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGF{beta}1 and PDGF{beta}{beta} stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and Smooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.\n\nResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGF{beta}1 and PDGF{beta}{beta} stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGF{beta}1 and PDGF{beta}{beta} stimulated PCLS and fibrotic PCLS.\n\nConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.