iPSC-derived organ-on-a-chip models for personalized human genetics and pharmacogenomics studies.

Genome-wide association studies (GWAS) have now correlated hundreds of genetic variants with complex genetic diseases and drug efficacy. Functional characterization of these factors remains challenging, particularly because of the lack of human model systems. Molecular and nanotechnological advances, in particular the ability to generate patient-specific PSC lines, differentiate them into diverse cell types, and seed and combine them on microfluidic chips, have led to the establishment of organ-on-a-chip (OoC) platforms that recapitulate organ biology. OoC technology thus provides unique personalized platforms for studying the effects of host genetics and environmental factors on organ physiology. In this review we describe the technology and provide examples of how OoCs may be used for disease modeling and pharmacogenetic research.

Construction of in vitro liver-on-a-chip models and application progress.

The liver is the largest internal organ of the human body. It has a complex structure and function and plays a vital role in drug metabolism. In recent decades, extensive research has aimed to develop in vitro models that can simulate liver function to demonstrate changes in the physiological and pathological environment of the liver. Animal models and in vitro cell models are common, but the data obtained from animal models lack relevance when applied to humans, while cell models have limited predictive ability for metabolism and toxicity in humans. Recent advancements in tissue engineering, biomaterials, chip technology, and 3D bioprinting have provided opportunities for further research in in vitro models. Among them, liver-on-a-Chip (LOC) technology has made significant achievements in reproducing the in vivo behavior, physiological microenvironment, and metabolism of cells and organs. In this review, we discuss the development of LOC and its research progress in liver diseases, hepatotoxicity tests, and drug screening, as well as chip combinations. First, we review the structure and the physiological function of the liver. Then, we introduce the LOC technology, including general concepts, preparation materials, and methods. Finally, we review the application of LOC in disease modeling, hepatotoxicity tests, drug screening, and chip combinations, as well as the future challenges and directions of LOC.

Liver-on-a-chip devices: the pros and cons of complexity.

Physiologically relevant and broadly applicable liver cell culture platforms are of great importance in both drug development and disease modeling. Organ-on-a-chip systems offer a promising alternative to conventional, static two-dimensional (2-D) cultures, providing much-needed cues such as perfusion, shear stress, and three-dimensional (3-D) cell-cell communication. However, such devices cover a broad range of complexity both in manufacture and in implementation. In this review, we summarize the key features of the human liver that should be reflected in a physiologically relevant liver-on-a-chip model. We also discuss different material properties of importance in producing liver-on-a-chip devices and summarize recent and current progress in the field, highlighting different types of devices at different levels of complexity.

Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET].

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF) and one of the leading indications for liver transplantation in Western societies. Given the wide use of both prescribed and over the counter drugs, DILI has become a major health issue for which there is a pressing need to find novel and effective therapies. Although significant progress has been made in understanding the molecular mechanisms underlying DILI, our incomplete knowledge of its pathogenesis and inability to predict DILI is largely due to both discordance between human and animal DILI in preclinical drug development and a lack of models that faithfully recapitulate complex pathophysiological features of human DILI. This is exemplified by the hepatotoxicity of acetaminophen (APAP) overdose, a major cause of ALF because of its extensive worldwide use as an analgesic. Despite intensive efforts utilising current animal and in vitro models, the mechanisms involved in the hepatotoxicity of APAP are still not fully understood. In this expert Consensus Statement, which is endorsed by the European Drug-Induced Liver Injury Network, we aim to facilitate and outline clinically impactful discoveries by detailing the requirements for more realistic human-based systems to assess hepatotoxicity and guide future drug safety testing. We present novel insights and discuss major players in APAP pathophysiology, and describe emerging in vitro and in vivo pre-clinical models, as well as advanced imaging and in silico technologies, which may improve prediction of clinical outcomes of DILI.

Hydrogel microfluidic-based liver-on-a-chip: Mimicking the mass transfer and structural features of liver.

Liver is fed by nutrition via diffusion across the vascular wall from blood flow. However, hepatocytes in liver models are directly exposed to the perfusion culture medium, where the shear stress reduces the cell viability and liver-specific functions. By mimicking the mass transfer and structural features of hepatic lobule, we designed a microfluidic liver-on-a-chip based on the di-acrylated pluronic F127 hydrogel. In the hydrogel chip, hepatocellular carcinoma HepG2 and human hepatic stellate cell LX-2 were statically cultured inside the microwells on the outer channel. These hepatic cells were fed by the diffused medium from the adjacent but separated inner channel with endothelial cell monolayers, which was perfused by the medium with physiologically relevant shear stress. As found, the hepatic cells in the liver-on-a-chip rapidly formed spheroids within 1-day incubation and expressed about one to two-fold higher viability/liver-specific functions than the corresponding static culture for at least 8 days. Moreover, the presence of endothelial cells also contributed to the expression of liver-specific functions in the liver-on-a-chip. Therefore, the proposed liver-on-a-chip provides a new concept for construction of 3D liver models in vitro, and shows the potential value for a variety of applications including bio-artificial livers and drug toxicity screening.

Quercetin and hydroxytyrosol as modulators of hepatic steatosis: A NAFLD-on-a-chip study.

Organs-on-chip (OoCs) are catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative. OoCs enable the creation of three-dimensional (3D) tissue microenvironments with physiological and pathological relevance at unparalleled precision and complexity, offering new opportunities to model human diseases and to test the potential therapeutic effect of drugs, while overcoming the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver-on-a-chip model to investigate the effects of two naturally occurring polyphenols, namely quercetin and hydroxytyrosol, on nonalcoholic fatty liver disease (NAFLD) using a high-content analysis readout methodology. NAFLD is currently the most common form of chronic liver disease; however, its complex pathogenesis is still far from being elucidated, and no definitive treatment has been established so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid-induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant and lipid-lowering properties. In conclusion, the findings of the present work could guide novel strategies to contrast the onset and progression of NAFLD.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro.

Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.

A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells.

Non-parenchymal cells play a key role in the occurrence and development of alcoholic liver disease. However, this cellular behaviour has not been fully characterized, and it is inconvenient to observe in traditional in vitro alcoholic liver disease (ALD) models and animal models. Herein we developed a demountable liver-on-chip device for investigation of pathophysiological process of individual non-parenchymal cells in alcohol induced ALD. This liver-device comprised of HepG2, LX-2, EAhy926 and U937 cells, which were ordered in a physiological distribution under perfuse. This device allows improved HepG2 cells activities and maintained high liver functions which including albumin synthesis and urea secretion. This novel liver-device is able to recreate the damage process of hepatic non-parenchymal cell lines induced by alcohol, and to understand the intercellular communication between different types of hepatic cells during ALD by measuring multiple biomarkers of each types of hepatic non-parenchymal cell lines, including Ve-cadherin, eNOS, VEGF and α-SMA. The proposed liver-device is able to further studies of pathological analysis and drug- and toxicity-screening.

Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications.

Maintenance of the complex phenotype of primary hepatocytes in vitro represents a limitation for developing liver support systems and reliable tools for biomedical research and drug screening. We herein aimed at developing a biosystem able to preserve human and rodent hepatocytes phenotype in vitro based on the main characteristics of the liver sinusoid: unique cellular architecture, endothelial biodynamic stimulation, and parenchymal zonation. Primary hepatocytes and liver sinusoidal endothelial cells (LSEC) were isolated from control and cirrhotic human or control rat livers and cultured in conventional in vitro platforms or within our liver-resembling device. Hepatocytes phenotype, function, and response to hepatotoxic drugs were analyzed. Results evidenced that mimicking the in vivo sinusoidal environment within our biosystem, primary human and rat hepatocytes cocultured with functional LSEC maintained morphology and showed high albumin and urea production, enhanced cytochrome P450 family 3 subfamily A member 4 (CYP3A4) activity, and maintained expression of hepatocyte nuclear factor 4 alpha (hnf4α) and transporters, showing delayed hepatocyte dedifferentiation. In addition, differentiated hepatocytes cultured within this liver-resembling device responded to acute treatment with known hepatotoxic drugs significantly different from those seen in conventional culture platforms. In conclusion, this study describes a new bioengineered device that mimics the human sinusoid in vitro, representing a novel method to study liver diseases and toxicology.

Microfluidic Gut-liver chip for reproducing the first pass metabolism.

After oral intake of drugs, drugs go through the first pass metabolism in the gut and the liver, which greatly affects the final outcome of the drugs' efficacy and side effects. The first pass metabolism is a complex process involving the gut and the liver tissue, with transport and reaction occurring simultaneously at various locations, which makes it difficult to be reproduced in vitro with conventional cell culture systems. In an effort to tackle this challenge, here we have developed a microfluidic gut-liver chip that can reproduce the dynamics of the first pass metabolism. The microfluidic chip consists of two separate layers for gut epithelial cells (Caco-2) and the liver cells (HepG2), and is designed so that drugs go through a sequential absorption in the gut chamber and metabolic reaction in the liver chamber. We fabricated the chip and showed that the two different cell lines can be successfully co-cultured on chip. When the two cells are cultured on chip, changes in the physiological function of Caco-2 and HepG2 cells were noted. The cytochrome P450 metabolic activity of both cells were significantly enhanced, and the absorptive property of Caco-2 cells on chip also changed in response to the presence of flow. Finally, first pass metabolism of a flavonoid, apigenin, was evaluated as a model compound, and co-culture of gut and liver cells on chip resulted in a metabolic profile that is closer to the reported profile than a monoculture of gut cells. This microfluidic gut-liver chip can potentially be a useful platform to study the complex first pass metabolism of drugs in vitro.

Long-term maintenance of a microfluidic 3D human liver sinusoid.

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPA's initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies.

Numerical investigation of dynamic microorgan devices as drug screening platforms. Part I: Macroscale modeling approach & validation.

The dynamic nature of in vitro drug metabolism models demands reliable numerical tools to determine key design parameter values towards high-fidelity cell-based platforms of in vivo drug metabolism. This paper represents the first of a two-part model-based investigation of a 3D dynamic microorgan device (DMD). The prescribed tissue model in this paper is precisely embedded within a DMD by 3D bioprinting hydrogel encapsulated liver cells into a patterned array of microchannels. A perfusing drug substrate is biotransformed by liver cells encapsulated within porous hydrogel walls. Therefore, the free and porous flow regime equations are first solved in tandem to derive the laminar velocity profile and wall shear stresses in the entire shear-mediated flow regime. These equations are then coupled with a convection-diffusion equation and Michaelis-Menten reaction terms, resulting in an effective convection-diffusion-cell kinetics model. A key consideration addressed herein is mechanotransduction where shear stresses on the encapsulated cells alter subcellular liver enzyme reaction rates. Cells are incorporated into the geometric model implicitly (macroscale) as enzyme reaction structures uniformly distributed throughout the DMD length. Transient simulations enable effluent drug metabolite profile determination wherein the proposed macroscale modeling approach is validated with an experimental drug flow study.

Numerical investigation of dynamic microorgan devices as drug screening platforms. Part II: Microscale modeling approach and validation.

The authors have previously reported a rigorous macroscale modeling approach for an in vitro 3D dynamic microorgan device (DMD). This paper represents the second of a two-part model-based investigation where the effect of microscale (single liver cell-level) shear-mediated mechanotransduction on drug biotransformation is deconstructed. Herein, each cell is explicitly incorporated into the geometric model as single compartmentalized metabolic structures. Each cell's metabolic activity is coupled with the microscale hydrodynamic Wall Shear Stress (WSS) simulated around the cell boundary through a semi-empirical polynomial function as an additional reaction term in the mass transfer equations. Guided by the macroscale model-based hydrodynamics, only 9 cells in 3 representative DMD domains are explicitly modeled. Dynamic and reaction similarity rules based on non-dimensionalization are invoked to correlate the numerical and empirical models, accounting for the substrate time scales. The proposed modeling approach addresses the key challenge of computational cost towards modeling complex large-scale DMD-type system with prohibitively high cell densities. Transient simulations are implemented to extract the drug metabolite profile with the microscale modeling approach validated with an experimental drug flow study. The results from the author's study demonstrate the preferred implementation of the microscale modeling approach over that of its macroscale counterpart.

Effect of Circulation Parameters on Functional Status of HepaRG Spheroids Cultured in Microbioreactor.

We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.

Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms.

We describe the generation of microfluidic platforms for the co-culture of primary hepatocytes and endothelial cells; these platforms mimic the architecture of a liver sinusoid. This paper describes a progressional study of creating such a liver sinusoid on a chip system. Primary rat hepatocytes (PRHs) were co-cultured with primary or established endothelial cells in layers in single and dual microchannel configurations with or without continuous perfusion. Cell viability and maintenance of hepatocyte functions were monitored and compared for diverse experimental conditions. When primary rat hepatocytes were co-cultured with immortalized bovine aortic endothelial cells (BAECs) in a dual microchannel with continuous perfusion, hepatocytes maintained their normal morphology and continued to produce urea for at least 30 days. In order to demonstrate the utility of our microfluidic liver sinusoid platform, we also performed an analysis of viral replication for the hepatotropic hepatitis B virus (HBV). HBV replication, as measured by the presence of cell-secreted HBV DNA, was successfully detected. We believe that our liver model closely mimics the in vivo liver sinusoid and supports long-term primary liver cell culture. This liver model could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, cancer research, and analysis of pathological effects and replication strategies of various hepatotropic infectious agents. .

Perfluoropolyether-Based Gut-Liver-on-a-Chip for the Evaluation of First-Pass Metabolism and Oral Bioavailability of Drugs.

In the evolving field of drug discovery and development, multiorgans-on-a-chip and microphysiological systems are gaining popularity owing to their ability to emulate in vivo biological environments. Among the various gut-liver-on-a-chip systems for studying oral drug absorption, the chip developed in this study stands out with two distinct features: incorporation of perfluoropolyether (PFPE) to effectively mitigate drug sorption and a unique enterohepatic single-passage system, which simplifies the analysis of first-pass metabolism and oral bioavailability. By introducing a bolus drug injection into the liver compartment, hepatic extraction alone could be evaluated, further enhancing our estimation of intestinal availability. In a study on midazolam (MDZ), PFPE-based chips showed more than 20-times the appearance of intact MDZ in the liver compartment effluent compared to PDMS-based counterparts. Notably, saturation of hepatic metabolism at higher concentrations was confirmed by observations when the dose was reduced from 200 µM to 10 µM. This result was further emphasized when the metabolism was significantly inhibited by the coadministration of ketoconazole. Our chip, which is designed to minimize the dead volume between the gut and liver compartments, is adept at sensitively observing the saturation of metabolism and the effect of inhibitors. Using genome-edited CYP3A4/UGT1A1-expressing Caco-2 cells, the estimates for intestinal and hepatic availabilities were 0.96 and 0.82, respectively; these values are higher than the known human in vivo values. Although the metabolic activity in each compartment can be further improved, this gut-liver-on-a-chip can not only be used to evaluate oral bioavailability but also to carry out individual assessment of both intestinal and hepatic availability.

Microfluidic Liver-on-a-Chip for Preclinical Drug Discovery.

Drug discovery is an expensive, long, and complex process, usually with a high degree of uncertainty. In order to improve the efficiency of drug development, effective methods are demanded to screen lead molecules and eliminate toxic compounds in the preclinical pipeline. Drug metabolism is crucial in determining the efficacy and potential side effects, mainly in the liver. Recently, the liver-on-a-chip (LoC) platform based on microfluidic technology has attracted widespread attention. LoC systems can be applied to predict drug metabolism and hepatotoxicity or to investigate PK/PD (pharmacokinetics/pharmacodynamics) performance when combined with other artificial organ-on-chips. This review discusses the liver physiological microenvironment simulated by LoC, especially the cell compositions and roles. We summarize the current methods of constructing LoC and the pharmacological and toxicological application of LoC in preclinical research. In conclusion, we also discussed the limitations of LoC in drug discovery and proposed a direction for improvement, which may provide an agenda for further research.

Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model.

OBJECTIVE: The liver acts as an innate immunity-dominant organ and natural killer (NK) cells, are the main lymphocyte population in the human liver. NK cells are in close interaction with other immune cells, acting as the first line of defense against pathogens, infections, and injury. A previously developed, three-dimensional, perfused liver-on-a-chip comprised of human cells was used to integrate NK cells, representing pivotal immune cells during liver injury and regeneration. The objective of this study was to integrate functional NK cells in an in vitro model of the human liver and assess utilization of the model for NK cell-dependent studies of liver inflammation. RESULTS: NK cells from human blood and liver specimen were isolated by Percoll separation with subsequent magnetic cell separation (MACS), yielding highly purified blood and liver derived NK cells. After stimulation with toll-like-receptor (TLR) agonists (lipopolysaccharides, Pam3CSK4), isolated NK cells showed increased interferon (IFN)-gamma secretion. To study the role of NK cells in a complex hepatic environment, these cells were integrated in the vascular compartment of a microfluidically supported liver-on-a-chip model in close interaction with endothelial and resident macrophages. Successful, functional integration of NK cells was verified by immunofluorescence staining (NKp46), flow cytometry analysis and TLR agonist-dependent secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. Lastly, we observed that inflammatory activation of NK cells in the liver-on-a-chip led to a loss of vascular barrier integrity. Overall, our data shows the first successful, functional integration of NK cells in a liver-on-a-chip model that can be utilized to investigate NK cell-dependent effects on liver inflammation in vitro.

Developing Liver Microphysiological Systems for Biomedical Applications.

Microphysiological systems (MPSs), also known as organ chips, are micro-units that integrate cells with diverse physical and biochemical environmental cues. In the field of liver MPSs, cellular components have advanced from simple planar cell cultures to more sophisticated 3D formations such as spheroids and organoids. Additionally, progress in microfluidic devices, bioprinting, engineering of matrix materials, and interdisciplinary technologies have significant promise for producing MPSs with biomimetic structures and functions. This review provides a comprehensive summary of biomimetic liver MPSs including their clinical applications and future developmental potential. First, the key components of liver MPSs, including the principal cell types and engineered structures utilized for cell cultivation, are briefly introduced. Subsequently, the biomedical applications of liver MPSs, including the creation of disease models, drug absorption, distribution, metabolism, excretion, and toxicity, are discussed. Finally, the challenges encountered by MPSs are summarized, and future research directions for their development are proposed.