Organ Model: Liver

Webinar Abstract

In vivo gene therapy holds enormous promise to treat inherited and acquired genetic diseases affecting the liver, including lysosomal storage disorders, liver metabolic disorders, and hemophilia. Creating an effective transgene for these genetic disorders is only half the battle—it’s just as critical to create a safe and efficient vehicle to deliver the transgene to the target cells.  

The development of these vehicles is where one of the biggest challenges lies and where progress has been hampered due to limitations of conventional research models. Animal studies are time consuming, costly, and tightly regulated, while 2D cell culture models lack the complexity to deliver physiological relevance. These challenges have led to a slow pace of gene therapy development for safe and effective therapeutics to become available for patients in need. 

Instead of waiting months to get results from animal studies that may not translate to humans, it is now possible to get physiologically relevant data within weeks by using the Emulate Liver-Chip to assess gene therapy vectors. The adeno-associated virus (AAV) transduction application for the Emulate Liver-Chip enables gene therapy researchers to test the delivery efficiency and toxicity of AAV vectors in the most human-relevant in vitro model of the liver sinusoid, with proven validity in predicting drug toxicity. This AAV transduction application will allow rapid iteration on AAV design to optimize the delivery of gene therapies and accelerate therapeutic development.

In this webinar, you will learn how the Liver-Chip has been used to: 

• Assess time- and concentration-dependent AAV transduction efficiency 
• Discriminate between the transduction efficiency of different AAVs 
• Evaluate the toxicity of AAV-based gene therapy vectors 
• Study AAV transport from vasculature to target epithelial tissue in a proof-of-concept study 

Overview

In this Application Note for adeno-associated virus (AAV) transduction, learn how researchers can test the delivery efficiency and safety of AAV vectors using the Emulate Liver-Chip.

Key highlights:

• Assess time- and concentration-dependent AAV transduction efficiency  
• Discriminate between the transduction efficiencies of different AAVs  
• Evaluate the toxicities of AAV-based gene therapy vectors  
• Study AAV transport from vasculature to target epithelial tissue in a proof-of-concept study  

Organ Model: Liver

Application: Toxicology

Abstract: Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.

Webinar Abstract

The liver is responsible for the internalization and catabolic clearance of therapeutic antibodies as well as antibody-bound immune complexes. Liver sinusoidal endothelial cells (LSECs) are key actors in these processes as they express scavenging receptors that recognize, bind, and internalize an enormous diversity of extracellular ligands.

Investigating the pharmacological effects of antibody clearance via human liver has been challenging to do in vitro due to, among other issues, the lack of reliable long-term LSEC culture protocols. In response to these issues, the Emulate Liver-Chip was developed as an effective in vitro model capable of elongating LSEC reliability, allowing scientists to study antibody clearance like never before.

In this webinar, you will:  

• Learn how the Emulate Liver-Chip was used to recreate the liver microenvironment and extend the viability and function of LSECs, including CD32B expression levels, for a duration relevant for assessing the pharmacokinetics (PK) of therapeutic antibodies.
• Hear about results that show that the expression of CD32B can differ based on experimental variables such as the source of primary cells (donor), passage number or source of detection antibodies used to visualize CD32B, and shear stress. 
• Understand how the CD32B expression was maintained for 14 days on the Liver-Chip in a donor-dependent but passage number independent manner and
• See how the Scanning Electron Microscopy (SEM) imaging showed the presence of fenestrae structures—one of the hallmarks of LSEC function. 

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 S1—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.