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

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  • March 19, 2019
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  • Norona, Leah
    • Affiliation: School of Medicine, Curriculum in Toxicology
Abstract
  • 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.
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  • In Copyright
Advisor
  • Coleman, William
  • Gerber, David
  • Otey, Carol
  • Watkins, Paul
  • Presnell, Sharon
  • Padilla, Stephanie
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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