A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices.

Hannah L Paish ,Abigail Watson,Jack Leslie,Steven A White,Derek A Mann,Gabriele Spoletini,Clive Griffiths,Jelena Mann,Mark C Bryan,Ben S Barksby,Jeremy French,Marco Y.W Zaki,Olivier Govaere,Helen Brown,Lee A Borthwick,Lee H Reed,Fiona Oakley,Julia C Whitehall ,Derek Manas ,Michael Drinnan ,Laura C Greaves ,Xin Xu ,Stuart Robinson ,Marina Garcia-Macía

doi:10.1002/hep.30651

Abstract

Precision cut liver slices (PCLSs) 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 aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme‐linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet‐derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α‐smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor‐cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell‐cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs 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 PCLSs without exogenous challenge. Activin receptor‐like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ‐stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor‐cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies.

Highlights

Hepatic fibrosis is characterized by accumulation of scar matrix in the liver and is the pathological consequence of persistent liver injury
We asked whether bidirectional flow was important and compared Precision cut liver slices (PCLSs) cultured in rocked BioR plates exposed to bidirectional flow, with PCLSs cultured under unidirectional flow, in a QuasiVivo circuit (Supporting Fig. S1B)
We describe a bioreactor that extends the health, metabolic activity, and functional longevity of PCLSs to at least 6 days and, importantly, without obvious hepatocellular stress and significant spontaneous fibrogenesis

Summary

Introduction

Hepatic fibrosis is characterized by accumulation of scar matrix in the liver and is the pathological consequence of persistent liver injury. ECM interactions found in the intact liver and are exposed to supraphysiological levels of mechanical stress when cultured directly on plastic The latter drastically alters cell behavior; for example quiescent hepatic stellate cells (qHSCs) grown on rigid tissue culture plastic transdifferentiate into α-smooth muscle actin (αSMA)+ HMs that express profibrotic genes and secrete ECM, whereas culturing qHSCs in soft Matrigel retains the features of HSC quiescence.[5]

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