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

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

Temporal Characterization of a Three-Dimensional Bioprinted Model Provides New Insight into Early Events Underlying Compound-Induced Fibrotic Liver Injury

Hepatic fibrosis develops from a series of complex and cumulative interactions among resident and recruited cells in response to sustained injury, making it a challenge to replicate using standard preclinical models. To understand early resident cell-mediated events that occur during this response, we took a three-dimensional approach using commercially available bioprinted liver tissues (ExVive3DTM Human Liver, Organovo) composed of primary human hepatocytes (HCs), endothelial cells (ECs), and hepatic stellate cells (HSCs). Because these cultures sustain important cell interactions and liver-specific functions over an extended period, we assessed the utility to recapitulate fundamental aspects of fibrogenesis following exposure to prototype fibrogenic agents. We first demonstrate compelling evidence of methotrexate-, TGF-β1-, and thioacetamide-induced fibrogenesis following two weeks of exposure with the rapid accumulation of collagen accompanied by transient cytokine release, HSC activation, and time-dependent upregulation of fibrosis-associated genes. To resolve early compound-induced effects, tissues were maintained post-manufacturing and allowed to reach steady-state cytokine production prior to dosing. Although tissue viability/function was not significantly altered, collagen deposition was attenuated suggesting the cytokine milieu post-manufacturing may influence the progression of the response. Temporal differences in LDH (general injury) and ALT (HC-specific injury) suggest HC injury precedes general, sustained injury following repeated methotrexate exposure. To understand the role of resident macrophages in modulating this response, Kupffer cells (KCs) were incorporated into the model. The pattern of general injury in the modified model suggests KCs shorten the injury window and reduce collagen deposition at the mid timepoint. These findings implicate the modulatory role of KCs during early exposure but suggest they may play a bimodal role during later phases where increased collagen deposition was observed. Because fibrosis is a dynamic response, recovery was also assessed. Monitoring of injury/functional markers following removal of the etiological agent suggests the model retains some biochemical capacity to recover. However, the two-week recovery timeframe may not have been sufficient to visualize collagen regression. This work lays the foundation for a well-defined, dynamic model of compound-induced liver fibrosis that will provide mechanistic insight into the early events underlying fibrogenesis and may inform the development of therapeutic strategies to prevent or reverse fibrosis.Doctor of Philosoph

Fig3

Collagen deposition in tissue sections from the standard (-KCs) and modified (+KCs) tissue model

Fig2

Urea and albumin secretion from the standard (-KCs) and modified (+KCs) tissue model

Fig1

LDH and miR-122 release from the standard (-KCs) and modified (+KCs) tissue model

Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis.

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents

Data from: Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 µM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents