Multiorgan-on-a-chip for realization of gut-skin axis.

The concept of physiological link between the gut and the skin, known as the gut-skin axis, has been gaining more evidence recently. Although experimental data from animal and human studies support the existence of the gut-skin axis, in vitro model platforms that can test the hypothesis are lacking. Organ-on-a-chip offers the possibility of connecting different tissues and recapitulating interactions between them. In this study, we report a multiorgan chip that can capture the basic interorgan communication between the gut and the skin. Its modular design enables separate culture and differentiation of the gut and skin tissues, and after assembly the two organs are connected via microfluidic channels than enables perfusion and mass transfer. We showed that the impairment of the gut barrier function exacerbated the adverse effect of fatty acids on skin cells, with decreased viability, increased level of cytokine secretion and human beta defensin-2 (hBD-2), an inflammatory dermal disease marker. Based on these results, we believe that our multiorgan chip can be a novel in vitro platform for recapitulating complex mechanisms underlying the gut-skin axis.

Organ-on-a-Chip for Studying Gut-Brain Interaction Mediated by Extracellular Vesicles in the Gut Microenvironment.

Extracellular vesicles (EVs) are a group of membrane vesicles that play important roles in cell-to-cell and interspecies/interkingdom communications by modulating the pathophysiological conditions of recipient cells. Recent evidence has implied their potential roles in the gut-brain axis (GBA), which is a complex bidirectional communication system between the gut environment and brain pathophysiology. Despite the evidence, the roles of EVs in the gut microenvironment in the GBA are less highlighted. Moreover, there are critical challenges in the current GBA models and analyzing techniques for EVs, which may hinder the research. Currently, advances in organ-on-a-chip (OOC) technologies have provided a promising solution. Here, we review the potential effects of EVs occurring in the gut environment on brain physiology and behavior and discuss how to apply OOCs to research the GBA mediated by EVs in the gut microenvironment.

Three-tissue microphysiological system for studying inflammatory responses in gut-liver Axis.

The interaction between the gut and the liver, often known as the gut-liver axis, play crucial roles in modulating the body's responses to the xenobiotics as well as progression of diseases. Dysfunction of the axis can cause metabolic disorders as well as obesity, diabetes, and fatty liver disease. During the progression of such diseases, inflammatory responses involving the immune system also play an important part. In this study, we developed a three-tissue microphysiological system (MPS) that can accommodate three different cell types in separated compartments connected via fluidic channels in a microfluidic device. Using computational fluid dynamics, geometry of fluidic channels and flow conditions were optimized for seeding and culturing different cell types in the three-tissue MPS. Caco-2 (gut), RAW264.7 (immune), and HepG2 (liver) cells were seeded and cultured in the chip. Stimulation of the gut cells in the MPS with lipopolysaccharide (LPS) resulted in induction of inflammatory response and production of nitric oxide (NO) in all connected chambers. The anti-inflammatory effect of luteolin was demonstrated. Our study demonstrates that the three-tissue MPS can recapitulate the inflammatory responses involving the gut, liver and immune cells.

Microfluidic skin chip with vasculature for recapitulating the immune response of the skin tissue.

There is a considerable need for cell-based in vitro skin models for studying dermatological diseases and testing cosmetic products, but current in vitro skin models lack physiological relevance compared to human skin tissue. For example, many dermatological disorders involve complex immune responses, but current skin models are not capable of recapitulating the phenomena. Previously, we reported development of a microfluidic skin chip with a vessel structure and vascular endothelial cells. In this study, we cocultured dermal fibroblasts and keratinocytes with vascular endothelial cells, human umbilical vascular endothelial cells. We verified the formation of a vascular endothelium in the presence of the dermis and epidermis layers by examining the expression of tissue-specific markers. As the vascular endothelium plays a critical role in the migration of leukocytes to inflammation sites, we incorporated leukocytes in the circulating media and attempted to mimic the migration of neutrophils in response to external stimuli. Increased secretion of cytokines and migration of neutrophils was observed when the skin chip was exposed to ultraviolet irradiation, showing that the microfluidic skin chip may be useful for studying the immune response of the human tissue.

Construction of pancreas-muscle-liver microphysiological system (MPS) for reproducing glucose metabolism.

Although in vitro models are widely accepted experimental platforms, their physiological relevance is often severely limited. The limitation of current in vitro models is strongly manifested in case of diseases where multiple organs are involved, such as diabetes and metabolic syndrome. Microphysiological systems (MPS), also known as organ-on-a-chip technology, enable a closer approximation of the human organs and tissues, by recreating the tissue microenvironment. Multiorgan MPS, also known as multiorgan-on-a-chip or body-on-a-chip, offer the possibility of reproducing interactions between organs by connecting different organ modules. Here, we designed a three-organ MPS consisting of pancreas, muscle, and liver, to recapitulate glucose metabolism and homeostasis by constructing a mathematical model of glucose metabolism, based on experimental measurement of glucose uptake by muscle cells and insulin secretion by pancreas cells. A mathematical model was used to modify the MPS to improve the physiological relevance, and by adding the liver model in the mathematical model, physiological realistic glucose and insulin profiles were obtained. Our study may provide a methodological framework for developing multiorgan MPS for recapitulating the complex interaction between multiple organs.

Gut-liver on a chip toward an in vitro model of hepatic steatosis.

Hepatic steatosis is a process of abnormal lipid deposition within the liver cells, often caused by excessive alcohol uptake or obesity. A conventional in vitro model for hepatic steatosis uses a liver cell culture, treated with fatty acids and measures accumulation of lipids within the cells. This model does not recapitulate the complex process of absorption and metabolism of digestive lipids. Here, we introduce a gut-liver chip, which mimics the gut absorption and hepatic metabolism in a microfluidic chip. Absorption of fatty acids through gut layer and subsequent deposition within liver cells was demonstrated. Tumor necrosis factor-α, butyrate, and α-lipoic acid were chosen as model molecules that can affect hepatic steatosis via different mechanisms, and their effects were evaluated. Our results suggest that the gut-liver chip can mimic the absorption and accumulation of fatty acids in the gut and the liver.

3D gut-liver chip with a PK model for prediction of first-pass metabolism.

Accurate prediction of first-pass metabolism is essential for improving the time and cost efficiency of drug development process. Here, we have developed a microfluidic gut-liver co-culture chip that aims to reproduce the first-pass metabolism of oral drugs. This chip consists of two separate layers for gut (Caco-2) and liver (HepG2) cell lines, where cells can be co-cultured in both 2D and 3D forms. Both cell lines were maintained well in the chip, verified by confocal microscopy and measurement of hepatic enzyme activity. We investigated the PK profile of paracetamol in the chip, and corresponding PK model was constructed, which was used to predict PK profiles for different chip design parameters. Simulation results implied that a larger absorption surface area and a higher metabolic capacity are required to reproduce the in vivo PK profile of paracetamol more accurately. Our study suggests the possibility of reproducing the human PK profile on a chip, contributing to accurate prediction of pharmacological effect of drugs.

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.

Microfluidic gut-on-a-chip with three-dimensional villi structure.

Current in vitro gut models lack physiological relevance, and various approaches have been taken to improve current cell culture models. For example, mimicking the three-dimensional (3D) tissue structure or fluidic environment has been shown to improve the physiological function of gut cells. Here, we incorporated a collagen scaffold that mimics the human intestinal villi into a microfluidic device, thus providing cells with both 3D tissue structure and fluidic shear. We hypothesized that the combined effect of 3D structure and fluidic shear may provide cells with adequate stimulus to induce further differentiation and improve physiological relevance. The physiological function of our '3D gut chip' was assessed by measuring the absorptive permeability of the gut epithelium and activity of representative enzymes, as well as morphological evaluation. Our results suggest that the combination of fluidic stimulus and 3D structure induces further improvement in gut functions. Our work provides insight into the effect of different tissue environment on gut cells.

Construction of 3D multicellular microfluidic chip for an in vitro skin model.

Current in vitro skin models do not recapitulate the complex architecture and functions of the skin tissue. In particular, on-chip construction of an in vitro model comprising the epidermis and dermis layer with vascular structure for mass transport has not been reported yet. In this study, we aim to develop a microfluidic, three-dimensional (3D) skin chip with fluidic channels using PDMS and hydrogels. Mass transport within the collagen hydrogel matrix was verified with fluorescent model molecules, and a transport-reaction model of oxygen and glucose inside the skin chip was developed to aid the design of the microfluidic skin chip. Comparison of viabilities of dermal fibroblasts and HaCaT cultured in the chip with various culture conditions revealed that the presence of flow plays a crucial role in maintaining the viability, and both cells were viable after 10 days of air exposure culture. Our 3D skin chip with vascular structures can be a valuable in vitro model for reproducing the interaction between different components of the skin tissue, and thus work as a more physiologically realistic platform for testing skin reaction to cosmetic products and drugs.

A pumpless multi-organ-on-a-chip (MOC) combined with a pharmacokinetic-pharmacodynamic (PK-PD) model.

A multi-organ-on-a-chip (MOC), also known as a human-on-a-chip, aims to simulate whole body response to drugs by connecting microscale cell cultures of multiple tissue types via fluidic channels and reproducing the interaction between them. While several studies have demonstrated the usefulness of MOC at a proof-of-concept level, improvements are needed to enable wider acceptance of such systems; ease of use for general biological researchers, and a mathematical framework to design and interpret the MOC systems. Here, we introduce a pumpless, user-friendly MOC which can be easily assembled and operated, and demonstrate the use of a PK-PD model for interpreting drug's action inside the MOC. The metabolism-dependent anticancer activity of a flavonoid, luteolin, was evaluated in a two-compartment MOC containing the liver (HepG2) and the tumor (HeLa) cells, and the observed anticancer activity was significantly weaker than that anticipated from a well plate study. Simulation of a PK-PD model revealed that simultaneous metabolism and tumor-killing actions likely resulted in a decreased anti-cancer effect. Our work demonstrates that the combined platform of mathematical PK-PD model and an experimental MOC can be a useful tool for gaining an insight into the mechanism of action of drugs with interactions between multiple organs. Biotechnol. Bioeng. 2017;114: 432-443. © 2016 Wiley Periodicals, Inc.

A microfluidic cell culture device (µFCCD) to culture epithelial cells with physiological and morphological properties that mimic those of the human intestine.

Physiological and morphological properties of the human intestine cannot be accurately mimicked in conventional culture devices such as well plates and petri dishes where intestinal epithelial cells form a monolayer with loose contacts among cells. Here, we report a novel microfluidic cell culture device (µFCCD) that can be used to culture cells as a human intestinal model. This device enables intestinal epithelial cells (Caco-2) to grow three-dimensionally on a porous membrane coated with fibronectin between two polydimethylsiloxane (PDMS) layers. Within 3 days, Caco-2 cells cultured in the µFCCD formed villi- and crypt-like structures with small intercellular spaces, while individual cells were tightly connected to one another through the expression of the tight junction protein occludin, and were covered with a secreted mucin, MUC-2. Caco-2 cells cultured in the µFCCD for 3 days were less susceptible to bacterial attack than those cultured in transwell plates for 21 days. µFCCD-cultured Caco-2 cells also displayed physiologically relevant absorption and paracellular transport properties. These results suggest that our intestinal model more accurately mimics the morphological and physiological properties of the intestine in vivo than the conventional transwell culture model.

Recent Advances in Gut- and Gut-Organ-Axis-on-a-Chip Models.

The human gut extracts nutrients from the diet while forming the largest barrier against the outer environment. In addition, the gut actively maintains homeostasis through intricate interactions with the gut microbes, the immune system, the enteric nervous system, and other organs. These interactions influence digestive health and, furthermore, play crucial roles in systemic health and disease. Given its primary role in absorbing and metabolizing orally administered drugs, there is significant interest in the development of preclinical in vitro model systems that can accurately emulate the intestine in vivo. A gut-on-a-chip system holds great potential as a testing and screening platform because of its ability to emulate the physiological aspects of in vivo tissues and expandability to incorporate and combine with other organs. This review aims to identify the key physiological features of the human gut that need to be incorporated to build more accurate preclinical models and highlights the recent progress in gut-on-a-chip systems and competing technologies toward building more physiologically relevant preclinical model systems. Furthermore, various efforts to construct multi-organ systems with the gut, called gut-organ-axis-on-a-chip models, are discussed. In vitro gut models with physiological relevance can provide valuable platforms for bridging the gap between preclinical and clinical studies.

Microfluidic gut-axis-on-a-chip models for pharmacokinetic-based disease models.

The low success rate of new drugs transitioning from animal testing to human clinical trials necessitates the development of more accurate and representative in vitro models. Recent advances in multi-organ-on-a-chip technology offer promising avenues for studying complex organ-organ interactions. Gut-liver-on-a-chip systems hold particular promise for mimicking the intricate interplay between the gut and liver, which play crucial roles in nutrient absorption, drug metabolism, detoxification, and immune response. Here, we discuss the key components of the gut-liver axis, including the gut epithelium, liver cells, gut microbiota, and their roles in the organ functions. We then explore the potential of gut-liver-on-a-chip models to replicate the intricate interactions between the two organs for pharmacokinetic studies and their expansion to more complicated multi-organ models. Finally, we provide perspectives and future directions for developing more physiologically relevant gut-liver-axis models for more efficient drug development, studying liver diseases, and personalizing treatment strategies.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

Multiplexing enhancement for the detection of multiple pathogen DNA.

Various methods for the detection of pathogens have been researched and developed. However, most of detection methods are cost-ineffective and laborious. To minimize costs and labor, multi-detection of pathogen have been widely used. In this paper, we propose a novel multi-detection method, which can enhance multi-detection capability using fluorescent dye labeled DNA nanostructures that is named DNA nanobarcodes. By using three fluorescence colors, multi-detection capability is significantly increased because of the increased combination of three colors. Moreover, our approach uses a relatively simple DNA nanostructure to precisely control the fluorescence intensity ratio. Therefore, high multi-detection ability is achieved without constructing a complicated DNA nanostructure probe. Our novel detection method can overcome the obstacles of conventional methods and enhance multi-detection capability effectively. By using our new system, we were able to successfully detect nine different DNA pathogens simultaneously. Our system can easily increase the multiplexibility by using more fluosrescent colors on DNA nanobarcode.

A microfluidic device with 3-d hydrogel villi scaffold to simulate intestinal absorption.

The absorption of drugs via oral route is a subject of a great interest in drug development process. The current in vitro method for measuring the kinetics of drug absorption relies on 2-D monolayer culture of Caco-2 cells on a porous membrane, but physiologically unrealistic environment provided by this method often results in inaccurate drug absorption kinetics. Here we report a novel microfluidic system which better mimics the physiological environment of the human small intestine. Three dimensional geometries of villi of the small intestine were reproduced via novel hydrogel microfabrication technique, and the fluid flow in the apical and basolateral sides of intestinal tract was reproduced with a two-layer microfluidic device. A wide range of flow rates was achieved by using gravity-induced flow, potentially facilitating easier high-throughput implementation. The kinetics of diffusion process through the 3-D villi scaffold in the microfluidic device was measured and mathematically modeled. When combined with intestinal cell culture model, this novel 3-D microfluidic system can serve as an in vitro platform that better mimics the in vivo environment.

Recent advances in in vitro skin-on-a-chip models for drug testing.

INTRODUCTION: The skin is an organ that has the largest surface area and provides a barrier against external environment. While providing protection, it also interacts with other organs in the body and has implications for various diseases. Development of physiologically realistic in vitro models of the skin in the context of the whole body is important for studying these diseases and will be a valuable tool for pharmaceutical, cosmetics, and food industry. AREA COVERED: This article provides an overview of the skin structure, physiology, as well as drug metabolism in the skin, and dermatological diseases. We summarize various in vitro skin models currently available, as well as novel in vitro models based on organ-on-a-chip technology. We also explain the concept of multi-organ-on-a-chip and describe recent developments in this field aimed at recapitulating the interaction of the skin with other organs in the body. EXPERT OPINION: Recent developments in the organ-on-a-chip field have enabled the development of in vitro model systems that resemble human skin more closely than conventional models. In the near future, we will be seeing various model systems that allow researchers to study complex diseases in a more mechanistic manner, which will help the development of new pharmaceuticals for such diseases.

On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices.

We describe a novel fabrication method that creates microporous, polymeric membranes that are either flat or contain controllable 3-dimensional shapes that, when populated with Caco-2 cells, mimic key aspects of the intestinal epithelium such as intestinal villi and tight junctions. The developed membranes can be integrated with microfluidic, multi-organ cell culture systems, providing access to both sides, apical and basolateral, of the 3D epithelial cell culture. Partial exposure of photoresist (SU-8) spun on silicon substrates creates flat membranes with micrometer-sized pores (0.5-4.0 µm) that--supported by posts--span across 50 µm deep microfluidic chambers that are 8 mm wide and 10 long. To create three-dimensional shapes the membranes were air dried over silicon pillars with aspect ratios of up to 4:1. Space that provides access to the underside of the shaped membranes can be created by isotropically etching the sacrificial silicon pillars with xenon difluoride. Depending on the size of the supporting posts and the pore sizes the overall porosity of the membranes ranged from 4.4 % to 25.3 %. The microfabricated membranes can be used for integrating barrier tissues such as the gastrointestinal tract epithelium, the lung epithelium, or other barrier tissues with multi-organ "body-on-a-chip" devices.