Translational Toxicology: Adapting Liver Microphysiological System for Cross-Species Evaluation of Hepatotoxicity Risk

Drug-induced liver injury (DILI) remains a leading cause of drug candidate attrition during development, underscoring the need for improved translational models that can predict human-specific toxicities. Traditional animal models, while informative for systemic toxicity, often fall short in forecasting human liver responses due to inherent interspecies differences. To address this translational gap, we leveraged the PhysioMimix® Organ-on-Chip (OOC) Core System to develop a robust DILI assay using primary hepatocytes from human, rat, and dog sources.
Methods
Using the PhysioMimix Core System, human, rat or dog hepatocytes were cultured as 3D liver microtissues under dynamic perfusion in the Liver-12 plate for up to 14 days, maintaining stable species-specific functionality (CYP activity, albumin, urea). A panel of six reference compounds (Troglitazone, Pioglitazone, Nefazodone, Buspirone, Tolcapone and Entacapone) recommended by the IQ MPS Consortium, including high-risk DILI agents and structural analogues with low human liability, was applied across a 7-point dosing regimen over four days. Functional in vitro and clinical biomarkers (albumin, urea, LDH, ALT) were monitored to assess hepatocellular health.
Results
The assay successfully captured known species-specific toxicities. For instance, nefazodone and troglitazone (DILI Rank 8) induced hepatotoxicity in human and rat models, while dog hepatocytes showed reduced sensitivity. Conversely, buspirone and entacapone exhibited minimal toxicity in animal models, though the human MPS detected subtle hepatotoxic signals at higher entacapone concentrations, highlighting its sensitivity to rare DILI events. Albumin consistently emerged as a sensitive cross-species marker, with dose-dependent decreases aligning with known toxic profiles.
Conclusions
These findings demonstrate the value of integrating human and animal liver MPS models to enhance cross-species interpretation and improve in vitro–in vivo extrapolation (IVIVE). The PhysioMimix system can be simply adapted to test multiple species in parallel to determine species-specific toxicity. As regulatory interest in MPS technologies grows, this approach offers a promising path to refine liver safety assessments, reduce reliance on and numbers of animal models used, and better predict human hepatotoxicity risks earlier in the drug development pipeline.

Presenter: Dr Emily Richardson

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Scaling Without Compromise: Liver MPS for Lead Optimization Screens to Investigative Toxicology

Drug-induced liver injury (DILI) remains the most common cause for acute liver failure in the USA and Europe and is a leading cause of attrition of compounds in drug development. Prediction of clinical safety is still a major hurdle in drug discovery and development, with many late-stage attrition events still occurring due to limited translatability of current in vitro or in vivo preclinical models. Microphysiological systems (MPS), otherwise known as organ-on-a-chip (OOC), provide a solution to this challenge by enabling the long-term culture and in-depth examination of highly human relevant liver microtissues. However, scaling and throughput of OOC have in the past been perceived as a challenge. Here, we present two solutions based on their different contexts of use. Firstly, a higher throughput approach, the PhysioMimix® Multi-chip Liver-48 plate can be applied within the lead optimization process for screening of compounds, up to 288 samples (or chips) per run. Secondly, due to its enhanced tissue size and sampling volume, the Liver-12 plate can further characterize DILI events at a deeper level and their associated mechanisms of toxicity through a wide range of liver-specific endpoints.
Methods
Using the PhysioMimix® Core System, primary human hepatocytes and Kupffer cells were cultured in 3D microtissues on engineered scaffolds in either the Liver-48 or Liver-12 plates under perfusion for eight days. The Liver-48 microtissues were subjected to 4-day exposure to two tool compounds recommended by the IQ MPS Consortium for DILI validation (troglitazone and pioglitazone) tested at seven concentrations alongside positive control (chlorpromazine) and vehicle control (0.1% DMSO). Each condition was tested in triplicate. For mechanistic studies, the Liver-12 microtissues were subjected to 10-day exposure to three tool compounds (diclofenac, troglitazone and pioglitazone). An equivalent dose to clinical concentration and 1.5x the Minimal Important Difference (MID) clinical concentration doses were tested alongside vehicle control (0.1% DMSO). Treatments were randomised across the plates and each condition tested in triplicate.
Results
Liver-48 plate microtissues were able to successfully differentiate the DILI risks of the tool compounds, using liver functionality (albumin, urea, ALT, AST) and viability (ATP) markers. The DILI profile of troglitazone was found to match those predicted by Liver-12 plate microtissues, demonstrating equivalence between the two plate types. The results of pioglitazone testing accurately predicted this compound to be safe. By assessing a range of test compounds recommended by the IQ MPS Consortium for DILI validation, the Liver-12 was able to correctly capture differing mechanisms of toxicity and mirror clinical outcomes. Oxidative stress, mitochondrial dysfunction, steatosis, dysregulation of bile acid synthesis or transport and inflammatory responses were investigated using a wide range of clinically relevant biomarkers.
Conclusions
OOC/MPS has long been considered a useful tool for better predicting human outcomes, however providing scalable throughput to meet lead optimization requirements and recoverable material at scale to enable biomarker detection sensitivity, plus deep mechanistic insights – has been a challenge. Here we demonstrate that the PhysioMimix Liver MPS can be scaled to enable specific contexts of use. By incorporating human-relevant insights into the earlier lead optimization phase of drug discovery using the Liver-48 plate facilitates the design of more refined pre-clinical studies. Conversely, when the context of use shifts from compounds screening to investigative toxicology, the Liver-12 DILI assay provides the optimal solution. Data-rich analyses from a wide range of clinically relevant biomarkers can be derived to make informed decisions to modify drug design and de-risk clinical progression of drug candidates. Together, this enables more efficient drug development and safer medicines in the future. Presenter:

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Recapitulating Immune-Driven Hepatotoxicity Using a Liver Microphysiological Platform

With the April 2025 U.S. Food and Drug Administration (FDA) roadmap promoting New Approach Methodologies (NAMs) for regulatory testing of monoclonal antibodies (mAbs) and other drugs, there is a need for models with greater complexity and human relevance. For biologics such as mAbs, immune-mediated drug-induced liver injury (iDILI) remains a significant concern in drug development. Human-specific immune system interactions with new modalities, such as therapeutic antibodies, can lead to unpredictable hepatotoxicity, which often fail to be captured in traditional animal models and high-throughput cell line models, leading to poor translational outcomes and high attrition rates. The FDA-recognised PhysioMimix® DILI assay was modified to incorporate peripheral blood mononuclear cells (PBMCs) to address this with an immune-competent, human liver relevant MPS model. The model aims to enhance translational relevance and improve safety profiling in drug development pipelines.
Methods
Primary human hepatocytes were cultured under dynamic perfusion in Liver-12 plates by PhysioMimix Core to form 3D liver microtissues. The liver microtissues were analysed for functionality (CYP activity, albumin, urea) and clinically relevant injury markers (ALT, AST). Following four days of hepatocyte culture, HLA-matched PBMCs were added to the circulating media with the liver microtissues for a further four days and functionality assessed (cytokine profiling). Ipilimumab (anti-CTLA-4) or infliximab (anti-TNF-alpha) was dosed into the media of hepatocyte only and co-cultures for 48 hours and samples were taken at 1-, 4-, 8-, 24- and 48-hours post-dosing. The liver microtissues were evaluated for hepatotoxic responses while immune activation and inflammation was assessed by cytokine release.
Results
Immune cell interaction and recovery from the Liver-12 was first completed to determine the ability of the PBMC to transverse the microfluidic channels and scaffolds unhindered (60-70% recovery). Following initiation of co-culture, maintenance of liver microtissue functionality and stable immune phenotypes was demonstrated over four days of co-culture. Upon dosing with the mAbs in the co-culture model, immune-mediated hepatotoxicity was detected with significant elevation in LDH and ALT/AST, and reduction in the functional biomarkers, albumin and urea. Interestingly, significant cytokine increases were only seen in ipilimumab-treated conditions, aligning with clinical data which demonstrated a pro-inflammatory phenotype via activation of T-cells.
Conclusion
This proof-of-concept study using two clinically relevant monoclonal antibodies demonstrates the ability of the PhysioMimix DILI assay to predict immune-mediated hepatotoxicity. Future studies will look to further profile immune alterations and interactions with the liver microtissues. Together, this assay provides a human-specific solution to the assessment of hepatotoxicity of large molecules. The FDA’s endorsement of NAMs for investigational new drug (IND) applications marks a paradigm shift toward human-relevant, non-animal testing strategies, positioning the PhysioMimix® Core System as a pivotal tool for the future of immunotoxicity assessment and safer drug development. Presenter: Dr. Justina Then

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