Dataset Information

Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.

ABSTRACT: The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce the in vitro formation of complex biliary networks using encapsulated immortalized mouse small biliary epithelial cells (cholangiocytes). This phenomenon is not observed using immortalized mouse large cholangiocytes, or with purified collagen 1 gels or Matrigel. We also show phenotypic stability via immunostaining for specific cholangiocyte markers. Moreover, tight junction formation and maturation was observed to occur between cholangiocytes, exhibiting polarization and transporter activity. To better define the mechanism of duct formation, we utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, radiating from a single nucleation point, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.

SUBMITTER: Lewis PL

PROVIDER: S-EPMC6093899 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.

Lewis Phillip L PL Su Jimmy J Yan Ming M Meng Fanyin F Glaser Shannon S SS Alpini Gianfranco D GD Green Richard M RM Sosa-Pineda Beatriz B Shah Ramille N RN

Scientific reports 20180815 1

The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce ...[more]

PMID: 30111800

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Project description:Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels.

Project description:Idiopathic pulmonary fibrosis is a chronic disease characterized by progressive lung scarring that inhibits gas exchange. Evidence suggests fibroblast-matrix interactions are a prominent driver of disease. However, available preclinical models limit our ability to study these interactions. We present a technique for synthesizing phototunable poly(ethylene glycol) (PEG)-based hybrid-hydrogels comprising healthy or fibrotic decellularized extracellular matrix (dECM) to decouple mechanical properties from composition and elucidate their roles in fibroblast activation. Here, we engineered and characterized phototunable hybrid-hydrogels using molecular techniques such as ninhydrin and Ellman's assays to assess dECM functionalization, and parallel-plate rheology to measure hydrogel mechanical properties. These biomaterials were employed to investigate the activation of fibroblasts from dual-transgenic Col1a1-GFP and αSMA-RFP reporter mice in response to changes in composition and mechanical properties. We show that reacting functionalized dECM from healthy or bleomycin-injured mouse lungs with PEG alpha-methacrylate (αMA) in an off-stoichiometry Michael-addition reaction created soft hydrogels mimicking a healthy lung elastic modulus (4.99 ± 0.98 kPa). Photoinitiated stiffening increased the material modulus to fibrotic values (11.48 ± 1.80 kPa). Percent activation of primary murine fibroblasts expressing Col1a1 and αSMA increased by approximately 40% following dynamic stiffening of both healthy and bleomycin hybrid-hydrogels. There were no significant differences between fibroblast activation on stiffened healthy versus stiffened bleomycin-injured hybrid-hydrogels. Phototunable hybrid-hydrogels provide an important platform for probing cell-matrix interactions and developing a deeper understanding of fibrotic activation in pulmonary fibrosis. Our results suggest that mechanical properties are a more significant contributor to fibroblast activation than biochemical composition within the scope of the hybrid-hydrogel platform evaluated in this study.Supplementary informationThe online version contains supplementary material available at 10.1007/s12195-022-00726-y.

Dataset Information

Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.

Publications

Complex bile duct network formation within liver decellularized extracellular matrix hydrogels.

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