Murine Precision-cut Intestinal Slices as a Potential Screening Tool for Antifibrotic Drugs.

BACKGROUND: Intestinal fibrosis is a hallmark of Crohn's disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. METHODS: Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1α1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor ß (TGF-ß) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. RESULTS: Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-ß1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1α1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. CONCLUSIONS: Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis.

Ex Vivo Model in Cholestasis Research.

To mimic (human) cholestasis in vitro requires multiple triggers to establish a diseased phenotype. However, this is currently not simulated by existing in vitro models. Therefore, there is a high need for multicellular systems similar to the human physiology. In such an in vitro model, cell-cell interactions and intact bile canaliculi with functional bile flow should be present and preserved during long-term culture. Precision-cut liver slices represent an ex vivo tissue culture technique that replicates most of the multicellular characteristics of a whole liver in vivo. This chapter describes the preparation and culturing of (human) precision-cut liver slices. Furthermore, a protocol to use the precision-cut liver slices technique to predict drug-induced cholestatic liver injury is described.

Peribiliary Glands Are Key in Regeneration of the Human Biliary Epithelium After Severe Bile Duct Injury.

Peribiliary glands (PBG) are a source of stem/progenitor cells organized in a cellular network encircling large bile ducts. Severe cholangiopathy with loss of luminal biliary epithelium has been proposed to activate PBG, resulting in cell proliferation and differentiation to restore biliary epithelial integrity. However, formal evidence for this concept in human livers is lacking. We therefore developed an ex vivo model using precision-cut slices of extrahepatic human bile ducts obtained from discarded donor livers, providing an intact anatomical organization of cell structures, to study spatiotemporal differentiation and migration of PBG cells after severe biliary injury. Postischemic bile duct slices were incubated in oxygenated culture medium for up to a week. At baseline, severe tissue injury was evident with loss of luminal epithelial lining and mural stroma necrosis. In contrast, PBG remained relatively well preserved and different reactions of PBG were noted, including PBG dilatation, cell proliferation, and maturation. Proliferation of PBG cells increased after 24 hours of oxygenated incubation, reaching a peak after 72 hours. Proliferation of PBG cells was paralleled by a reduction in PBG apoptosis and differentiation from a primitive and pluripotent (homeobox protein Nanog+/ sex-determining region Y-box 9+) to a mature (cystic fibrosis transmembrane conductance regulator+/secretin receptor+) and activated phenotype (increased expression of hypoxia-inducible factor 1 alpha, glucose transporter 1, and vascular endothelial growth factor A). Migration of proliferating PBG cells in our ex vivo model was unorganized, but resulted in generation of epithelial monolayers at stromal surfaces. Conclusion: Human PBG contain biliary progenitor cells and are able to respond to bile duct epithelial loss with proliferation, differentiation, and maturation to restore epithelial integrity. The ex vivo spatiotemporal behavior of human PBG cells provides evidence for a pivotal role of PBG in biliary regeneration after severe injury.