Differential fibrotic phenotypes of hepatic stellate cells within 3D liver organoids

Abstract

Liver fibrosis occurs in most cases of chronic liver disease, which are somewhat common, but also a potentially deadly group of diseases. In vitro modeling of liver fibrosis relies primarily on the isolation of in vivo activated hepatic stellate cells (aHSCs) and studying them in standard tissue culture dishes (two‐dimensional [2D]). In contrast, modeling of fibrosis in a biofabricated three‐dimensional (3D) construct allows us to study changes to the environment, such as extracellular matrix (ECM) composition and structure, and tissue rigidity. In the current study, we used aHSCs produced through subcultures in 2D and encapsulated them in a 3D collagen gel to form spherical constructs. In parallel, and as a comparison, we used an established HSC line, LX‐2, representing early and less severe fibrosis. Compared with LX‐2 cells, the aHSCs created a stiffer environment and expressed higher levels of TIMP1 and LOXL2, all of which are indicative of advanced liver fibrosis. Collectively, this study presents a fibrosis model that could be incorporated with multi‐cellular models to more accurately reflect the effects of a severe fibrotic environment on liver function.

Biofabricated 3D in vitro model of fibrosis‐induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver

“…HepaRG+aHSC organoids showed fibers significantly longer and thicker than within HepaRG+LX-2 organoids ( Figure 3B). In a previous study we showed that a monoculture of aHSC cells created longer and thicker fibers than the monoculture of LX-2 cells [36]. Similar analysis of collagen fibers were carried out in healthy and cirrhotic liver specimens.…”

“…factors (TGF-β) associated with both fibrosis and development (Supplemental Figure 3), addition of ALK5i effectively reduced TGF-β gene expression [36].…”

Chemokine Therapy in Cats With Experimental Renal Fibrosis and in a Kidney Disease Pilot Study

“…Irreversible fibrosis results when the tissue microenvironment favors ECM production and deposition over degradation and remodeling. Analysis of collagen fiber parameters, such as length, width, angularity, and straightness, in normal and pathologic disease states may help unlock the conditions that favor irreversible fibrosis ( 40 , 43 , 44 , 46 ), and this method of evaluating collagen has been validated by Caetano et al as comparable to the gold standard hydroxyproline assay ( 45 ). Increased collagen fiber width in the tumor microenvironment is a powerful negative prognostic indicator associated with reduced 5-year survival that was validated in two separate gastric cancer cohorts of 225 and 151 cancer patients ( 42 ).…”

“…The results of 10 images were averaged for each cat ( N = 30) for further analysis. For tissues stained with Picrosirius Red under polarized light (PSR-POL), two collagen fiber analyses were performed: (1) Collagen fiber quantification for length and width using the segmentation software CT-FIRE (Laboratory for Optical and Computation Instrumentation, University of Wisconsin) ( 39 – 45 ) ( Figure 4B ); and (2) Hue analysis for level of collagen bundling and total collagen quantification using a MATLAB script (R2020a, MathWorks, Natick, MA) ( 43 , 44 , 46 ). Results of 10 images were averaged for each cat ( N = 30) for further analysis.…”

In Vitro Three‐Dimensional Liver Models for Nanomaterial DNA Damage Assessment

“…Multiple models have been developed in an attempt to recreate a pro‐fibrotic environment using the introduction of hepatic stellate cells, which have been acknowledged as the leading scar forming cell type in most liver injuries. [ 32,37 ] It has been frequently found that the greater presence of hepatic stellate cells is commonly linked to the proliferation of hepatoma cells and enhanced tumor metastasis. [ 38,39 ]…”