Organ Model: Liver

Abstract

Background and Aims: Nonalcoholic fatty liver disease (NAFLD) is a progressive condition initially characterized by increased lipid accumulation in the liver (steatosis) and can develop into nonalcoholic steatohepatitis (NASH). There is an unmet need for a human-relevant in vitro model to enable successful development of therapies. 

Methods: To address this unmet need, we utilized our human Liver-Chip, which retains key characteristics of native liver function over long-term culture. To induce steatosis, chips were treated with saturated (palmitate) or unsaturated (oleate) fatty acids, alone or in combination. TGF-beta was used as a positive control for hepatocellular injury and stellate cell activation. To assess therapeutic efficacy against steatosis, chips were treated for two days after initiating steatosis (therapeutic), or co-treated (prophylactic) with a liver-targeted analogue of firsocostat, a known inhibitor of acetyl-CoA carboxylase (ACC-i). Morphological evaluation of the hepatocytes and AdipoRed^TM staining was used to evaluate steatosis. Quantification of triglycerides released in the media was used to evaluate lipid removal, and alpha-SMA staining was used to assess stellate cell activation. 

Results: We demonstrated induction of steatosis in hepatocytes in a concentration-dependent manner following continuous exposure to oleate, palmitate, or in combination. Withdrawal of fatty acids significantly diminished the steatotic phenotype as well as levels of triglycerides released in accordance with relevant human in vivo data. Administration of TGF-beta resulted in increased stellate cell activation, hepatocellular injury, and lipid accumulation compared to the vehicle controls. Chips treated with the ACC-i demonstrated a concentration-dependent reduction in lipid accumulation in both the therapeutic and prophylactic paradigms when compared to steatosis induced controls.

Conclusions: In this study we provide preliminary data supporting the potential application of the Liver-Chip for modeling NAFLD-like phenotypes and conducting human-relevant therapeutic efficacy assessment using clinically relevant endpoints.

Abstract

Drug-induced liver injury (DILI) remains a major cause of drug attrition during drug discovery and development because animal models and existing in vitro models often do not predict outcome in humans. There is a significant need for more predictive models for DILI. These models must include the relevant cell types that are representative of in vivo metabolic capabilities, provide the ability to conduct long-term maintenance of cell viability to enable repeated drug exposures, and include the capability to demonstrate the diverse mechanisms of DILI. Advanced engineering fabrication techniques were applied to achieve a high level of control over the liver tissue microenvironment. The Liver-Chip incorporates relevant cell-extra cellular matrix (ECM) interactions, a hepatocyte and liver sinusoidal endothelial cell interface, along with relevant cyto-architecture and physiological flow. In addition to the human Liver-Chip, rat and dog models were developed to enable characterization of species differences with respect to pharmacokinetics, toxicity, and mechanism of action. 

Abstract

The pharmaceutical industry has an unmet need for predictive human models for drug metabolism and pharmacokinetics, drug-drug interactions, and drug-induced liver injury (DILI) that can better emulate human response to drugs. Current 2D liver models often fail to capture responses seen in the clinic, as the cellular microenvironment does not accurately reflect what is found in vivo. Here we applied a vascularized human Liver-Chip model that contains primary human hepatocytes, sinusoidal endothelial cells, and Kupffer cells, cultured under physiological fluid flow in a spatial configuration that recapitulates their cytoarchitecture in the liver resulting in long-term viability and improved functionality. The Organ-Chip’s fluidic structure allows for easy effluent sampling for the detection of various biomarkers and cytokine release, and the optical clarity of the Organ-Chip allows for various morphological analyses throughout the culture duration. This provides a platform to investigate mechanistic insights of various DILI and demonstrates the value of the human Liver-Chips for predicting human metabolism, safety testing, risk assessment, and drug discovery and development.

Abstract

Liver lipid accumulation (fatty liver) is an early step in the development of drug-induced, alcoholic (ASH) and non-alcoholic steatohepatits (NASH) that may progress into cirrhosis and hepatocellular carcinoma.

A wealth of animal studies and in vitro models have elucidated molecular mechanisms associated with NASH, whereas translation of these findings to effective therapeutics for humans, remains an unmet medical need. In the effort to establish more predictive and human-relevant models for fatty liver disease/steatosis, a microengineered Liver-Chip was developed to include hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer, and stellate cells for more accurate representation of the in vivo tissue. The Liver-Chip has the potential for long-term maintenance of cell viability  to enable repeated drug exposure and exhibited hepatic functions that recapitulate in vivo metabolic capabilities. The Liver-Chip has well-characterized toxicology endpoints sensitive for cell-specificity over time and is capable of demonstrating the diverse mechanisms of drug-induced liver injury. The model incorporates relevant cell-ECM interactions, a hepatocyte and LSEC interface, with relevant cytoarchitecture and physiological flow. Liver-specific functions were measured for all three species and demonstrated maintenance of in vivo relevant levels of functionality including albumin secretion, CYP450 enzyme activity, and gene  expression of certain markers that demonstrated improved function over conventional monolayer models.  We evaluated the ability of the human Liver-Chip to respond to steatosis inducers (e.g., free fatty acid, glucose and ethanol).

Overview

An analysis of 150 drug candidates that caused adverse events in humans demonstrated that testing in rats and dogs only predicted 71% of toxicities in humans. The liver is of particular concern, as drug-induced liver injury (DILI) is the primary cause for withdrawal of drugs from the market and throughout the clinical development process.

Our Liver-Chip can help address this translational challenge by recreating in vivo-relevant liver function in a more physiological microenvironment than conventional cell-based models.

This application note reviews how researchers can use our Liver-Chip to:

• Predict diverse mechanisms of unexpected human toxicity for preclinical drug candidates
• Assess the human relevance of toxicity observed in preclinical animal studies
• Translate mechanism of toxicity for candidates with unexpected safety signals in the clinic

 Overview

In the pharmaceutical industry, there is a need for more human-relevant preclinical models that can enable a mechanistic understanding of drug action.

Organ-Chips can help address this need by recreating in vivo-relevant function in a more physiological microenvironment than conventional cell-based models.

In this technical note, we review how our Chip-S1 can be used to create a comprehensive recapitulation of the liver sinusoid.

Key highlights:

• Liver-Chip emulates in vivo-like physiology by incorporating all key hepatic cell types in the sinusoid
• Continuous perfusion of media ensures cells are exposed to sufficient amounts of test compounds, metabolites, or other stimulus factors
• Cells can be fixed and imaged for further analysis or removed and processed for genomic studies
• Potential applications include safety testing, mechanistic toxicity, disease modeling, mechanism of action determination, and biomarker identification

Organ Model: Liver

Application: ADME-Tox

Abstract: Microphysiological systems (MPS) are emerging as potentially predictive models for drug safety and toxicity assessment. To assess the utility of these systems, the Food and Drug Administration partnered with Emulate to evaluate the Human Liver Organ-Chip in a regulatory setting. Diglycolic acid (DGA), a known hepatotoxin, was evaluated in the Liver-Chip and compared to a multi-well plate format to assess the Liver-Chip’s capabilities, limitations, overall performance, and concordance with other in vivo and in vitro studies. Cryopreserved primary human hepatocytes were exposed to DGA from 1 to 20 mM in Liver-Chips or traditional multi-well plates. We found that 10 mM or 20 mM of DGA was severely cytotoxic in both platforms, while 5 mM was mildly cytotoxic in Liver-Chips. Additionally, some hepatocyte functions were reduced with 5 mM DGA in Liver-Chips and 1 mM in well plates. Individual well effects were greater or occurred sooner than in the Liver-Chips. Examination of the performance of the Liver-Chip showed that variability was low for biochemical endpoints, but higher for imaging endpoints. Sensitivity and specificity were high. Only 3-4 Liver-Chips were necessary to detect an effect depending on the endpoint and effect size. The specifics of the experiment are found herein.

Spotting species-specific toxicity

Candidate drug testing using standard preclinical models cannot accurately predict which compounds are likely to cause drug-induced liver injury in humans. To improve selection of promising drug candidates, Jang et al. developed a Liver-Chip consisting of rat, dog, or human hepatocytes, endothelial cells, Kupffer cells, and stellate cells. Using the microfluidic chips, the authors confirmed mechanism of action of several known hepatotoxic drugs and an experimental compound. A second experimental compound that induced fibrosis in a rat Liver-Chip did not alter hepatocyte function in human chips, whereas a third compound demonstrated increased toxicity in a dog Liver-Chip. Results support using multispecies chips to identify species-specific differences in drug metabolism and toxicity.

Abstract

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

Published in: Alternatives to Animal Experimentation

Abstract

Investigative toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, investigative toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adherence to the 3R (Replacement, Reduction, and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, investigative toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of investigative toxicology is to improve pre-clinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded owing to the use of inappropriate animal models. Progress in investigative toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field, a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive, and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an “Investigative Toxicology Think Tank”, which aimed to enhance interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights investigative toxicology’s position by identifying key challenges and perspectives.

Organ Model: Liver

Application: Toxicology

Abstract: Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose- and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC₅₀ 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC₅₀ 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC₅₀ 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.