Co-Culture of Human Primary Hepatocytes and Nonparenchymal Liver Cells in the Emulate® Liver-Chip for the Study of Drug-Induced Liver Injury.

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.

Modeling alcohol-associated liver disease in a human Liver-Chip.

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.

Reproducing human and cross-species drug toxicities using a Liver-Chip.

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.

Introducing an automated high content confocal imaging approach for Organs-on-Chips.

Organ-Chips are micro-engineered systems that aim to recapitulate the organ microenvironment. Implementation of Organ-Chips within the pharmaceutical industry aims to improve the probability of success of drugs reaching late stage clinical trial by generating models for drug discovery that are of human origin and have disease relevance. We are adopting the use of Organ-Chips for enhancing pre-clinical efficacy and toxicity evaluation and prediction. Whilst capturing cellular phenotype via imaging in response to drug exposure is a useful readout in these models, application has been limited due to difficulties in imaging the chips at scale. Here we created an end-to-end, automated workflow to capture and analyse confocal images of multicellular Organ-Chips to assess detailed cellular phenotype across large batches of chips. By automating this process, we not only reduced acquisition time, but we also minimised process variability and user bias. This enabled us to establish, for the first time, a framework of statistical best practice for Organ-Chip imaging, creating the capability of using Organ-Chips and imaging for routine testing in drug discovery applications that rely on quantitative image data for decision making. We tested our approach using benzbromarone, whose mechanism of toxicity has been linked to mitochondrial damage with subsequent induction of apoptosis and necrosis, and staurosporine, a tool inducer of apoptosis. We also applied this workflow to assess the hepatotoxic effect of an active AstraZeneca drug candidate illustrating its applicability in drug safety assessment beyond testing tool compounds. Finally, we have demonstrated that this approach could be adapted to Organ-Chips of different shapes and sizes through application to a Kidney-Chip.