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

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.

Figure 1

Generation of PCLSs and optimization of the bioreactor culture method. (A) Workflow used to generate PCLSs. (B) Photographs show the BioR plate containing six bioreactor chamber units and a zoomed image of one chamber unit, which contains two wells connected by a channel. (C) Photograph of PCLSs cultured on 8‐μm Transwell inserts in the BioR plate. (D,E) Graphs show secreted albumin (ng/mL) levels in media of PCLSs cultured on 3‐ or 8‐μm Transwell inserts in either a rocking BioR plate (D) or static 12‐well Transwell plate (E).

Figure 2

Modeling fibrosis and testing antifibrotic therapy in normal rat liver slices. (A) Graph shows soluble Col1a1 (ng/mL) levels in media of bioreactor‐cultured rPCLSs after 24‐hour rest and then 24‐ and 72‐hour culture ± fib stim (TGFβ1/PDGFββ) ± Alk5i (48‐ and 96‐hour total culture). (B‐D) mRNA levels of Col1A1, αSMA, and TIMP1 in rPCLSs at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± Alk5i (96‐hour total culture). (A‐D) Numbers on bars show fold change compared to t = 0. (E) Representative 100× magnification images of αSMA and picrosirius red–stained rPCLSs from three different livers at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± Alk5i. Scale bars = 200 µm. (F) Graph shows percentage area of picrosirius red–stained tissue in rPCLS at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± Alk5i. Data are mean ± SEM in n = 4 different livers. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001. Abbreviations: Cont, control; fib stim, fibrotic stimulation; IHC, immunohistochemistry; Veh, vehicle.

Figure 3

Bioreactor‐cultured fibrotic rat liver slices can be maintained for 6 days. (A) Graph shows hydroxyproline (μg/g of liver tissue) in bioreactor‐cultured fPCLSs. (B‐D) mRNA expression of Col1A1, αSMA, and TIMP1 in bioreactor‐cultured fPCLSs at t = 0 and 2‐, 3‐, 4‐, and 6‐day culture. Data are mean ± SEM in n = 5 independent slice experiments. (E) Representative images of αSMA and picrosirius red–stained rat fPCLS at t = 0 and 2, 3, 4, and 6 days in culture. Scale bars = 200 µm. *P < 0.05; **P < 0.01. Abbreviation: IHC, immunohistochemistry.

Figure 4

Bioreactor‐cultured human liver slices retain ductular cells, endothelial cells, and macrophages. Representative images show CK19‐stained biliary epithelial cells (left), CD31‐stained endothelial cells (middle), and CD68‐stained macrophages (right) in t = 0 and days 1, 2, 3, 4, and 6 cultured hPCLS. CK19 and CD31 images are at 400× magnification and CD68 are at 200× magnification.

Figure 5

Modeling fibrosis and testing antifibrotic therapy in human liver slices using soluble markers. (A‐H) Graphs show fibronectin (μg/mL), HA (ng/mL), TIMP1 (ng/mL), MMP1 (ng/mL), MMP7 (ng/mL), MMP10 (ng/mL), IL‐6 (pg/mL), IL‐1β (pg/mL), and VEGF (pg/mL) levels in media of bioreactor‐cultured hPCLSs after 24‐hour rest and then 24‐ or 72‐hour culture ± fib stim (TGFβ1/PDGFββ) ± Alk5i; numbers on bars show fold change compared to 48‐hour control. Data are mean ± SEM in n = 4 independent slice experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Abbreviations: Cont, control; fib stim, fibrotic stimulation; Veh, vehicle.

Figure 6

Modeling fibrosis and testing antifibrotic therapy in human liver slices using gene expression and histology. (A) mRNA levels of Col1A1, αSMA, and TIMP1 in hPCLS at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± Alk5i; numbers on bars show fold change compared to t = 0. (B) Representative 100× magnification images of αSMA and (C) picrosirius red–stained hPCLSs from three different donor livers at t = 0 and after 24‐h rest + 72‐hour culture (4 days) ± fib stim ± Alk5i. Scale bars = 200 µm. (D) Representative 200× magnification image of picrosirius red–stained hPCLSs from donor 4. (E) Percentage area of picrosirius red–stained tissue in hPCLSs at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± Alk5i (96‐hour total culture). Data are mean ± SEM in n = 4 independent slice experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Abbreviations: Cont, control; fib stim, fibrotic stimulation; IHC, immunohistochemistry; Veh, vehicle.

Figure 7

Assessing impact antifibrotic therapies in fibrosis‐induced rat liver slices. (A‐C) mRNA levels of Col1A1, αSMA, and TIMP1 in rPCLS at t = 0 and after 24‐hour rest + 72‐hour culture ± fib stim ± nintedanib, OCA, and losartan (96‐hour total culture). Data are mean ± SEM in n = 5 different livers. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001. (D) Average media albumin (ng/mL) levels in bioreactor‐cultured rPCLSs after 24‐hour rest and then 24‐ to 72‐hour culture ± fib stim (TGFβ1/PDGFββ) ± Alk5i, nintedanib, OCA, and losartan (48‐ and 96‐hour total culture). Abbreviations: Cont, control; fib stim, fibrotic stimulation; Nint, nintedanib; Veh, vehicle.