Organ Model: Liver

Webinar Abstract

In this webinar, Dr. Jonathan Sexton presents an evaluation of iPSC-derived human liver organoids (HLOs) that spontaneously produce autologous hepatocytes, stellate, and Kupffer cells for drug-induced liver injury (DILI) risk prediction. The study leveraged Emulate Organ-Chips while using a multi-omics approach that integrated metabolomics, single-cell RNA sequencing, and high-content imaging to predict DILI risk with imputation of the mechanism of action. HLOs on the Liver-Chip were shown to dramatically increase albumin production and CYP450 expression while releasing ALT/AST when treated with drugs known to cause DILI at clinically relevant concentrations. Furthermore, HLO Liver-Chips were able to be used to evaluate inarigivir for hepatitis B by predicting the hepatotoxicity of the tenofovir-inarigivir combination that was responsible for unanticipated liver injury and death in a phase-III clinical study. This combination caused steatosis and mitochondrial perturbation in HLOs that recapitulate the clinical and histological presentation of the liver injury with a mechanism similar to fialuradine. 

The study “A Multi-Omics Human Liver Organoid Screening Platform for DILI Risk Prediction” is available to read on bioRxiv.

OVERVIEW

The Liver-Chip S1 BioKit includes the essential components needed to create the Liver-Chip—including Emulate qualified cells—and is available in multiple sizes to meet various study needs.

Webinar Abstract

This is webinar is presented on data from a bioRxiv preprint.  The final version of this paper, “Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology” is now live in Communications Medicine, part of Nature Portfolio.

To hear commentary from the authors about the impact these findings could have on the future of research and drug development, watch our webinar “Towards A More Predictive Model of Human Biology: A Fireside Chat.”

Failure in late stages of the drug development pipeline is one of the major challenges the pharmaceutical industry faces today. Human organ-on-a-chip (Organ-Chip) technology has the potential to disrupt preclinical drug discovery, as it has been shown to recapitulate organ-level pathophysiology and clinical responses. Additionally, industrial guidelines have been published that describe the criteria for qualifying preclinical models for a particular use application; however, systematic and quantitative evaluation of Organ-Chips’ predictive value has not been conducted to date.

To explore how this challenge might be approached, 780 human Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury caused by small molecules. Across a blinded set of 27 known hepatotoxic and non-toxic drugs, the LIVER-CHIP demonstrated a sensitivity of 87% and a specificity of 100%. A computational economic value analysis suggests that, with this performance, the Liver-Chip could generate $3 billion per year to small-molecule drug development by driving an increase in research and development productivity. 

In this webinar, we discuss: 

• Why preclinical models with greater predictive validity will improve clinical success and productivity

• How the Emulate Liver-Chip performed against the IQ MPS guidelines

• How the Emulate Liver-Chip compared to animal models and hepatic spheroids

• What the economic impact of the Liver-Chip in routine use of small-molecule liver toxicity could be

• Where the Emulate Liver-Chip can be implemented into the drug development process

Toxicological Sciences (2021)

Abstract

New approach methodologies (NAMs), including in vitro toxicology methods such as human cells from simple cell cultures to 3D and organ-on-a-chip models of human lung, intestine, liver, and other organs, are challenging the traditional “norm” of current regulatory risk assessments. Uncertainty Factors continue to be used by regulatory agencies to account for perceived deficits in toxicology data. With the expanded use of human cell NAMs, the question “Are uncertainty factors needed when human cells are used?” becomes a key topic in the development of 21st-century regulatory risk assessment. Michael Dourson, Ph.D., the co-author of a paper detailing uncertainty factors within the US EPA, and Lorna Ewart, Ph.D., Executive Vice President, Science, Emulate, who is involved in developing organ-on-a-chip models, debated the topic

One important outcome of the debate was that in the case of in vitro human cells on a chip, the interspecies (animal to human) uncertainty factor of 10 could be eliminated. However, in the case of the intraspecies (average human to sensitive human), the uncertainty factor of 10, additional toxicokinetic and/or toxicodynamic data or related information will be needed to reduce much less eliminate this factor. In the case of other currently used uncertainty factors, such as LOAEL to NOAEL extrapolation, missing important toxicity studies, and acute/subchronic to chronic exposure extrapolation, additional data might be needed even when using in vitro human cells. Collaboration between traditional risk assessors with decades of experience with in vivo data and risk assessors working with modern technologies like organ chips is needed to find a way forward.

Organ Model: Liver

Application: Immunology & Inflammation

Abstract: Alcohol-associated liver disease (ALD) is a global health issue and leads to progressive liver injury, comorbidities, and increased mortality. Human-relevant preclinical models of ALD are urgently needed. Here, we leverage a triculture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi to model ALD employing human-relevant blood alcohol concentrations (BACs) and multimodal profiling of clinically relevant endpoints. Our Liver-Chip recapitulates established ALD markers in response to 48 h of exposure to ethanol, including lipid accumulation and oxidative stress, in a concentration-dependent manner and supports the study of secondary insults, such as high blood endotoxin levels. We show that remodeling of the bile canalicular network can provide an in vitro quantitative readout of alcoholic liver toxicity. In summary, we report the development of a human ALD Liver-Chip as a powerful platform for modeling alcohol-induced liver injury with the potential for direct translation to clinical research and evaluation of patient-specific responses.

Overview

Purpose: 

Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD) are a growing public health concern¹, especially in the United States where it affects one quarter of the adult population². The accumulation of excess fat in the liver causes progressive cell damage and inflammation which in severe forms of steatohepatitis can be toxic and lead to end-stage liver disease. Due to the lack of human relevant preclinical disease models to test lead candidate drugs, there are no clinically approved therapies targeting these diseases. To overcome this, we are developing a model for studying alcoholic steatosis using the Human Liver-Chip to induce cytotoxic levels of lipid accumulation by treating with increasing concentrations of ethanol, an important mediator of disease pathogenesis in AFLD patients. 

Methods: 

Human Quad-Culture Liver-Chips (n=3 for each condition) were coated with a mixture of rat tail collagen type I and bovine fibronectin. Primary human hepatocytes from two donors (HU8305 and CYC) were seeded at a density of 3.5 million cells/mL in the upper parenchymal channel and later overlaid with Matrigel and incubated at 37°C with 5% CO₂.  In the lower vascular channel on the opposite side of the porous membrane, human liver sinusoidal endothelial cells (LSECs) (3 million cells/mL), human liver Kupffer (0.5 million cells/mL) and stellate cells (0.1 million cells/mL) were seeded. Two days later, the Chips were connected to Zoë^® Culture Module (Human Emulation System^®), and both the Chip channels were perfused at a constant flow of 30 µL/h. On Day 7 post seeding, the Liver-Chips were treated with ethanol at 0.16% or 0.5% (v/v). The Chips were maintained for 11 days with imaging and effluent collection on days 1, 3, 7, 10 and 11. On Day 11 post-treatment, the experiment was terminated, and Chips were fixed for immunofluorescent imaging with AdipoRed™ for lipid droplet accumulation and DAPI in the top channel and α-Smooth muscle actin (SMA) for activated stellate cells and DAPI in the bottom channel. 

Results:

A severe time- and concentration-dependent toxic response was observed in both hepatocyte donors, post ethanol treatment. Qualitatively, the ethanol treated groups demonstrated a concentration-dependent increase in lipid droplet accumulation and activated stellate cells, in the HU8305 hepatocytes indicative of cell damage. Similarly, the ethanol treated groups demonstrated an increase in lipid accumulation in the CYC hepatocytes, with similar stellate cell activation between the control and ethanol treated groups in this donor. Additionally, the 0.5% ethanol treated group showed a significant decrease in albumin release and an increase in ALT and triglyceride export in the CYC donor suggesting cytotoxicity from Day 7 post-treatment. 

Conclusion: 

The Human Quad-Culture Liver-Chip model demonstrated a time- and concentration-dependent increase in intracellular hepatic lipid accumulation (steatosis) following ethanol treatment indicative of toxicity.  Activation of hepatic stellate cells, albumin secretion, ALT release and triglyceride export were also affected by ethanol treatment along with variability between the donors. Thus, based on the promising preliminary results from this study, further investigation is needed to enable evaluation of therapeutic agent efficacy in this Liver-Chip model of alcohol induced steatosis.

References: 

1 Fazel et al.,Epidemiology and natural history of non-alcoholic fatty liver disease. Metabolism. 2016 Aug;65(8):1017-25. doi: 10.1016/j.metabol.2016.01.012. Epub 2016 Jan 29. PMID: 26997539.
2 Perumpail et al., Clinical epidemiology and disease burden of nonalcoholic fatty liver disease, World J Gastroenterol. 2017 Dec 21; 23(47): 8263–8276.

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).