Microfabrication of human organs-on-chips
TL;DR: A protocol for the fabrication, microengineering and operation of microfluidic organ-on-chip systems that replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro is described and can be easily adapted to develop other human organ chips.
Abstract: 'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.
Citations
More filters
TL;DR: The aim of this article is to review the different TEER measurement techniques and analyze their strengths and weaknesses, determine the significance of TEER in drug toxicity studies, and examine the various in vitro models and microfluidic organs-on-chips implementations using TEER measurements in some widely studied barrier models.
Abstract: Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood–brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article ...
1,300 citations
TL;DR: 2D and 3D cell culture methods are reviewed, advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes are discussed, and directions for future research are suggested.
Abstract: Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.
1,048 citations
TL;DR: This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria and proof-of-principle to show that the microfluidic gut-on-a-chip device can be used to create human intestinal disease models and gain new insights into gut pathophysiology.
Abstract: A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.
662 citations
Cites methods from "Microfabrication of human organs-on..."
...The gut-on-a-chip microdevice used in this study was fabricated from PDMS as reported previously (12, 16, 33)....
[...]
...The coculture method in the gut-on-a-chip described here enables stable host–microbe coexistence because of the presence of continuous fluid flow and peristalsis-like motions that enhance intestinal differentiation (33) and permit bacterial populations to reach a dynamic steady-state (12) that can be sustained over weeks in culture....
[...]
TL;DR: This Review examines how tissue barrier properties, parenchymal tissue function and multi-organ interactions can be recreated in organ-on-a-chip systems and applied for drug screening.
Abstract: Predicting the effects of drugs before human clinical trials is at the heart of drug screening and discovery processes. The cost of drug discovery is steadily increasing owing to the limited predictability of 2D cell culture and animal models. The convergence of microfabrication and tissue engineering gave rise to organ-on-a-chip technologies, which offer an alternative to conventional preclinical models for drug screening. Organ-on-a-chip devices can replicate key aspects of human physiology crucial for the understanding of drug effects, improving preclinical safety and efficacy testing. In this Review, we discuss how organ-on-a-chip technologies can recreate functions of organs, focusing on tissue barrier properties, parenchymal tissue function and multi-organ interactions, which are three key aspects of human physiology. Specific organ-on-a-chip systems are examined in terms of cell sources, functional hallmarks and available disease models. Finally, we highlight the challenges that need to be overcome for the clinical translation of organ-on-a-chip devices regarding materials, cellular fidelity, multiplexing, sensing, scalability and validation. Organ-on-a-chip devices can recreate key aspects of human physiology in vitro, offering an alternative to animal models for preclinical drug testing. This Review examines how tissue barrier properties, parenchymal tissue function and multi-organ interactions can be recreated in organ-on-a-chip systems and applied for drug screening.
624 citations
TL;DR: E engineered in vitro models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer are provided.
Abstract: The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.
445 citations
References
More filters
TL;DR: Mechanically active “organ-on-a-chip” microdevices that reconstitute tissue-tissue interfaces critical to organ function may expand the capabilities of cell culture models and provide low-cost alternatives to animal and clinical studies for drug screening and toxicology applications.
Abstract: Here, we describe a biomimetic microsystem that reconstitutes the critical functional alveolar-capillary interface of the human lung. This bioinspired microdevice reproduces complex integrated organ-level responses to bacteria and inflammatory cytokines introduced into the alveolar space. In nanotoxicology studies, this lung mimic revealed that cyclic mechanical strain accentuates toxic and inflammatory responses of the lung to silica nanoparticles. Mechanical strain also enhances epithelial and endothelial uptake of nanoparticulates and stimulates their transport into the underlying microvascular channel. Similar effects of physiological breathing on nanoparticle absorption are observed in whole mouse lung. Mechanically active "organ-on-a-chip" microdevices that reconstitute tissue-tissue interfaces critical to organ function may therefore expand the capabilities of cell culture models and provide low-cost alternatives to animal and clinical studies for drug screening and toxicology applications.
3,081 citations
TL;DR: New advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microen environments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs are reviewed.
1,501 citations
TL;DR: This gut-on-a-chip recapitulates multiple dynamic physical and functional features of human intestine that are critical for its function within a controlled microfluidic environment that is amenable for transport, absorption, and toxicity studies, and hence it should have great value for drug testing as well as development of novel intestinal disease models.
Abstract: Development of an in vitro living cell-based model of the intestine that mimics the mechanical, structural, absorptive, transport and pathophysiological properties of the human gut along with its crucial microbial symbionts could accelerate pharmaceutical development, and potentially replace animal testing. Here, we describe a biomimetic ‘human gut-on-a-chip’ microdevice composed of two microfluidic channels separated by a porous flexible membrane coated with extracellular matrix (ECM) and lined by human intestinal epithelial (Caco-2) cells that mimics the complex structure and physiology of living intestine. The gut microenvironment is recreated by flowing fluid at a low rate (30 μL h−1) producing low shear stress (0.02 dyne cm−2) over the microchannels, and by exerting cyclic strain (10%; 0.15 Hz) that mimics physiological peristaltic motions. Under these conditions, a columnar epithelium develops that polarizes rapidly, spontaneously grows into folds that recapitulate the structure of intestinal villi, and forms a high integrity barrier to small molecules that better mimics whole intestine than cells in cultured in static Transwell models. In addition, a normal intestinal microbe (Lactobacillus rhamnosus GG) can be successfully co-cultured for extended periods (>1 week) on the luminal surface of the cultured epithelium without compromising epithelial cell viability, and this actually improves barrier function as previously observed in humans. Thus, this gut-on-a-chip recapitulates multiple dynamic physical and functional features of human intestine that are critical for its function within a controlled microfluidic environment that is amenable for transport, absorption, and toxicity studies, and hence it should have great value for drug testing as well as development of novel intestinal disease models.
1,247 citations
TL;DR: This work presents a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks and demonstrates utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins.
Abstract: Tissue function depends on hierarchical structures extending from single cells ( approximately 10 microm) to functional subunits (100 microm-1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved and have the advantages of miniaturization. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'.
1,085 citations
TL;DR: This work discusses PDMS absorption and its potential impact on microfluidic experiments.
Abstract: Microfluidic devices made out of polydimethylsiloxane (PDMS) have many physical properties that are useful for cell culture applications, such as transparency and gas permeability. Another distinct characteristic of PDMS is its ability to absorb hydrophobic small molecules. Partitioning of molecules into PDMS can significantly change solution concentrations and could potentially alter experimental outcomes. Herein we discuss PDMS absorption and its potential impact on microfluidic experiments.
944 citations