Editor's Highlight: Modeling Compound-Induced Fibrogenesis In Vitro Using Three-Dimensional Bioprinted Human Liver Tissues

Abstract

Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate- and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1β) during the early culture time period is followed by concentration- and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-β1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment.

Keywords: 3D bioprinted liver; compound-induced liver injury; liver fibrosis in vitro.

FIG. 1

3D bioprinted tissue recapitulates the tissue-like density and architecture of normal liver. (A) Transverse cross-sections of native human liver and (B) bioprinted human liver tissue stained with H&E. (C and D) Formation of hepatocellular junctions is shown with E-cadherin and the mesenchymal marker (E and F) vimentin is used to highlight distribution patterns within the parenchyma analogous to native liver. Scale bar = 50 µm.

FIG. 2

3D bioprinted tissue exhibits a compartmentalized architecture and maintains hepatic stellate cells in a quiescent-like phenotype. (A) Illustration of a transverse cross-section of bioprinted tissue on a transwell insert comprising hepatocytes (HCs) and compartmentalized endothelial cells (ECs) and hepatic stellate cells (HSCs). (B) The organization of non-parenchymal cells (NPCs) is depicted with CD31 and vimentin staining to mark ECs and HSCs, respectively. Albumin is used to denote the hepatocellular compartment (HC). Scale bar = 100 µm, inset scale bar = 25 µm. (C) HSC activation status was examined using desmin (generic marker) and α-SMA (activation marker). Quiescent HSCs are denoted with white arrows. Scale bar = 50 µm.

FIG. 3

Impact of fibrotic agents on biochemical markers of liver tissue viability and functionality. (A) Gross images of tissues following 14 days of treatment. Scale bar = 2.5 mm. (B and C) LDH release during an extended 14-day treatment with MTX, TAA, and TGF-β1 (n = 9 for Tx1–Tx7, n = 5 for Tx9–Tx14). (D) Albumin production as a measure of hepatocellular function is depicted at key time points during the treatment period (n = 5). Significance was determined using a one-way ANOVA with post hoc Dunnett’s multiple comparisons test (*P < .05, **P < .01, ***P < .001, ****P < .0001).

FIG. 4

H&E and trichrome staining reveals key features consistent with clinical fibrosis in bioprinted tissues following 14 days of treatment with select fibrogenic agents. Representative sections of bioprinted liver treated with (A) 0.1% DMSO vehicle, (B) 0.1 and (C) 10 ng/mL TGF-β1, (F) 0.1 and (G) 1.0 µM MTX, and (D) 5.0 and (E) 25 mM TAA. (B and C) A circle is used to delineate the non-parenchymal (NPC) from the parenchymal (HC) compartments and white arrows denote the basolateral edge of the tissue in contact with the transwell membrane. Collagen deposition was visualized (blue) in successive sections of bioprinted tissue stained with Gomori’s trichrome. Entrapped hepatocytes (EH), nodular areas of collagen deposition (NF), pericellular fibrosis (PF; F and G inset, 150% enlarged), (G) yellow arrows denote bridging fibrosis. Scale bar = 100 µm. (F) Expression of fibrosis-associated genes at Tx7 and Tx14 in MTX-treated tissue.

FIG. 5

Increased deposition of collagens I and IV and expression of vimentin and α-SMA in tissues exhibiting pronounced fibrogenic change. Tissues treated with 0.1% DMSO vehicle, 1.0 µM MTX, and 25 mM TAA were further assessed immunohistochemically for (A) collagen I, (B) collagen IV, (C) vimentin, and (D) α-SMA. White and yellow arrows denote the apical and basolateral edges of the tissue, respectively. Areas of fibrillar ECM deposition are outlined in successive collagen I and collagen IV stained sections. Nodular areas of collagen deposition (NF). The percent area covered by collagens I and IV is depicted in the bottom left-hand corner of the photomicrographs. (C) Punctate areas of vimentin positivity in control tissue (white arrows) and diffuse patterning in treated tissue (yellow arrows). The black and white inset accentuates the shift in vimentin patterning observed with treatment. (D) α-SMA(+) HSCs were mainly noted at the periphery of the tissue in the vehicle-treated control (white arrows). Increased SMA(+) HSCs in the center of treated tissues and altered distribution of the cells corresponding to areas of collagen deposition (white arrows). Scale bar = 25 µm.

FIG. 6

Subset of cytokines exhibiting treatment-dependent differences over time and at select treatment time points. (A) The upregulation (red) and downregulation (green) of proinflammatory, immunoregulatory, and chemotactic cytokines relative to vehicle-treated control was represented in a heat map. (B) Samples collected at mid (Tx7) and late (Tx14) treatment time points were profiled for additional cytokines and chemokines. Values outside the range of the standard curve or excluded via Grubb’s outlier analysis are shaded grey.