Coculture model of a liver sinusoidal endothelial cell barrier and HepG2/C3a spheroids-on-chip in an advanced fluidic platform.

The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier's permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using acetaminophen, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of acetaminophen by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.

Investigation of the metabolomic crosstalk between liver sinusoidal endothelial cells and hepatocytes exposed to paracetamol using organ-on-chip technology.

Organ-on-chip technology is a promising in vitro approach recapitulating human physiology for the study of responses to drug exposure. Organ-on-chip cell cultures have paved new grounds for testing and understanding metabolic dose-responses when evaluating pharmaceutical and environmental toxicity. Here, we present a metabolomic investigation of a coculture of liver sinusoidal endothelial cells (LSECs, SK-HEP-1) with hepatocytes (HepG2/C3a) using advanced organ-on-chip technology. To reproduce the physiology of the sinusoidal barrier, LSECs were separated from hepatocytes by a membrane (culture insert integrated organ-on-chip platform). The tissues were exposed to acetaminophen (APAP), an analgesic drug widely used as a xenobiotic model in liver and HepG2/C3a studies. The differences between the SK-HEP-1, HepG2/C3a monocultures and SK-HEP-1/HepG2/C3a cocultures, treated or not with APAP, were identified from metabolomic profiles using supervised multivariate analysis. The pathway enrichment coupled with metabolite analysis of the corresponding metabolic fingerprints contributed to extracting the specificity of each type of culture and condition. In addition, we analysed the responses to APAP treatment by mapping the signatures with significant modulation of the biological processes of the SK-HEP-1 APAP, HepG2/C3a APAP and SK-HEP-1/HepG2/C3a APAP conditions. Furthermore, our model shows how the presence of the LSECs barrier and APAP first pass can modify the metabolism of HepG2/C3a. Altogether, this study demonstrates the potential of a "metabolomic-on-chip" strategy for pharmaco-metabolomic applications predicting individual response to drugs.

Liver organ-on-chip models for toxicity studies and risk assessment.

The liver is a key organ that plays a pivotal role in metabolism and ensures a variety of functions in the body, including homeostasis, synthesis of essential components, nutrient storage, and detoxification. As the centre of metabolism for exogenous molecules, the liver is continuously exposed to a wide range of compounds, such as drugs, pesticides, and environmental pollutants. Most of these compounds can cause hepatotoxicity and lead to severe and irreversible liver damage. To study the effects of chemicals and drugs on the liver, most commonly, animal models or in vitro 2D cell cultures are used. However, data obtained from animal models lose their relevance when extrapolated to the human metabolic situation and pose ethical concerns, while 2D static cultures are poorly predictive of human in vivo metabolism and toxicity. As a result, there is a widespread need to develop relevant in vitro liver models for toxicology studies. In recent years, progress in tissue engineering, biomaterials, microfabrication, and cell biology has created opportunities for more relevant in vitro models for toxicology studies. Of these models, the liver organ-on-chip (OoC) has shown promising results by reproducing the in vivo behaviour of the cell/organ or a group of organs, the controlled physiological micro-environment, and in vivo cellular metabolic responses. In this review, we discuss the development of liver organ-on-chip technology and its use in toxicity studies. First, we introduce the physiology of the liver and summarize the traditional experimental models for toxicity studies. We then present liver OoC technology, including the general concept, materials used, cell sources, and different approaches. We review the prominent liver OoC and multi-OoC integrating the liver for drug and chemical toxicity studies. Finally, we conclude with the future challenges and directions for developing or improving liver OoC models.

Development of Liver-on-Chip Integrating a Hydroscaffold Mimicking the Liver's Extracellular Matrix.

The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study.

Validation of HepG2/C3A Cell Cultures in Cyclic Olefin Copolymer Based Microfluidic Bioreactors.

Organ-on-chip (OoC) technology is one of the most promising in vitro tools to replace the traditional animal experiment-based paradigms of risk assessment. However, the use of OoC in drug discovery and toxicity studies remain still limited by the low capacity for high-throughput production and the incompatibility with standard laboratory equipment. Moreover, polydimethylsiloxanes, the material of choice for OoC, has several drawbacks, particularly the high absorption of drugs and chemicals. In this work, we report the development of a microfluidic device, using a process adapted for mass production, to culture liver cell line in dynamic conditions. The device, made of cyclic olefin copolymers, was manufactured by injection moulding and integrates Luer lock connectors compatible with standard medical and laboratory instruments. Then, the COC device was used for culturing HepG2/C3a cells. The functionality and behaviour of cultures were assessed by albumin secretion, cell proliferation, viability and actin cytoskeleton development. The cells in COC device proliferated well and remained functional for 9 days of culture. Furthermore, HepG2/C3a cells in the COC biochips showed similar behaviour to cells in PDMS biochips. The present study provides a proof-of-concept for the use of COC biochip in liver cells culture and illustrate their potential to develop OoC.