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Airborne toxicological assessment: The potential of lung-on-a-chip as an alternative to animal testing
Abstract
Recent studies have shown that there exists a direct relationship between environmental pollutants (PM2.5, smog), the respiratory system, and the morbidity and mortality of cardiovascular diseases. However, the mechanism and principle of how these harmful substances are deposited in lung tissues and impair lung function remain unclear. It is important to gain improved understanding of the interaction between environmental pollutants and human lungs. Owing to the complexity of air pollution and toxicological risks, it is difficult to predict and evaluate the response of human lungs toward the damage caused by air pollution. Although animal models can be used as a basis for toxicological classification, the toxic effect on the human body could be very different from that on animals owing to the distinctive features of different species. This article provides a comprehensive review of in vitro lung-on-a-chip technologies and their application in the toxicological assessment of environmental pollutants. A lung-on-a-chip uses a bionic structure mimicking the physiological characteristic of lungs, features of a real airway, and condition of the physiological airflow. Accordingly, it can be used to reveal the intrinsic interaction between lung tissues and particulate matter and provide new insights into the effect of the toxicology of environmental particles on lungs. In addition, the development of novel and optimized lung-on-a-chip devices and their application devices in the health assessment of air pollution are expected to overcome the limitations of the current in vitro toxicological tests. They are also anticipated to provide effective and accurate methods for drug screening and toxicity testing. Finally, the application potential of in vitro lung-on-a-chip models is emphasized in this review.
... The chip is a microfluidic device with networks of hair-fine microchannels for managing solution volumes ranging from picoliters to milliliters. The organ refers to microscopic tissues created on microfluidic chips that may simulate the functions of distinct organs (Donkers et al., 2021;Lin et al., 2022). Although these systems are simpler than genuine human tissues and organs, they may replicate human physiology and disease. ...
... Numerous surgeries have been extended owing to hematopoietic stem cell toxicity (Aziz et al., 2019;Musah et al., 2017). Due to their rapid proliferation and renewal, hematopoietic stem cells (HSCs) in the bone marrow may be harmed by chemical agents that direct their progenitors toward mature cells (Arjmand et al., 2022;Fritschen and Blaeser, 2021;Lin et al., 2022). Previously, preclinical toxicity research was conducted using a variety of laboratory animals, which had a number of disadvantages (Balijepalli and Sivaramakrishan, 2017;Vulto and Joore, 2021). ...
... However, by putting three-dimensional cell cultures onto these devices, their biological complexity is increased (Paloschi et al., 2021). By manipulating the microenvironment and grown cell types, important disease traits may be reproduced (Aziz et al., 2019;Lin et al., 2022). On-chip models of cardiac ischemia, fibrosis, and cardiotoxicity are among the heart-on-chip systems that have been reported (Owaki et al., 2013;Richards et al., 2020). ...
The core of the drug research and screening processes is predicting the effect of drugs prior to human clinical trials. Due to the 2D cell culture and animal models' poor predictability, the cost of drug discovery is continuously rising. The development of organ-on-a-chip technology, an alternative to traditional preclinical drug testing models, resulted from the intersection of microfabrication & tissue engineering. Preclinical safety and effectiveness testing is improved by the ability of organ-on-a-chip technologies to mimic important human physiological functions necessary for understanding drug effects. Organ-on-a-chip could drastically improve the success rate of the preclinical testing thereby better predicting how the drug will act on the clinical trials. Organ-on-a-chip is a term used to describe a microengineered biomimetic device that mimics the structure and functionality of human tissue. It integrates engineering, cell biology, & biomaterial technologies on a miniature platform. To reflect human physiology in vitro and bridge the gap between in vivo and in vitro data, simplification shouldn't compromise physiological relevance. At this level of organ-on-a-chip technological development, biomedical engineers specializing in device engineering are more important than ever to expedite the transfer of technology from the academic lab bench to specialized product development institutions and an ever-growing market. This review focuses on the recent advancements in the organ-on-a-chip technology and discusses the potential of this technology based on the current available literature.
... Air pollution has become a major environmental hazard factor affecting global health (Almetwally et al., 2020;Singh et al., 2021b). Inhalation is the main route of atmospheric pollutants exposure and inhalant exposure to pollutants has serious health consequences such as respiratory disease, lung cancer, gastrointestinal disorders, neurological disorders, liver illnesses and cardiovascular disease (Hu et al., 2022;Lin et al., 2022;Kang et al., 2021b;Vignal et al., 2021). It is a hot topic worthy of high attention to assess the potential risks of atmospheric pollutants, including particulate matters (PMs), nanoparticles (NPs), bioaerosols, and some chemicals, on human health during pulmonary exposure (Singh et al., 2021a). ...
... However, the lung is a complex and sophisticated organ with multiple cell types, extracellular matrix (ECM) components, mechanical cues and multi-organs interactions, thus 2D cell culture is difficult to simulate key features and functions of lung organ due to the lack of dynamic physiological environment of the lung (Sen et al., 2022;Polaka et al., 2022). The defects of traditional models limit further understanding of how atmospheric pollutants deposit in and/or pass through the alveolar-capillary barrier to affect the biological functions of lung, as well as other organs (Lin et al., 2022;Sznitman, 2022). Thus, there is necessary to develop new models to represent the features of lung and assess the health risks of atmospheric pollutants exposure in respiratory systems. ...
Atmospheric pollutants, including particulate matters, nanoparticles, bioaerosols, and some chemicals, have posed serious threats to the environment and the human's health. The lungs are the responsible organs for providing the interface betweenthecirculatory system and the external environment, where pollutant particles can deposit or penetrate into bloodstream circulation. Conventional studies to decipher the mechanismunderlying air pollution and human health are quite limited, due to the lack of reliable models that can reproduce in vivo features of lung tissues after pollutants exposure. In the past decade, advanced near-to-native lung chips, combining cell biology with bioengineered technology, present a new strategy for atmospheric pollutants assessment and narrow the gap between 2D cell culture and in vivo animal models. In this review, the key features of artificial lung chips and the cutting-edge technologies of the lung chip manufacture are introduced. The recent progresses of lung chip technologies for atmospheric pollutants exposure assessment are summarized and highlighted. We further discuss the current challenges and the future opportunities of the development of advanced lung chips and their potential utilities in atmospheric pollutants associated toxicity testing and drug screening.
... Despite the progress, challenges remain in accurately delivering particles in in-vivo models, and the limitations of conventional in-vitro models to mimic realistic physiochemical conditions hinder further advancements in this research domain [6][7][8]. The development of lung-on-achip technology in 2010 offered a novel approach to address these challenges, realistically replicating the air-blood barrier of the lungs within a microchannel and allowing for direct exposure of cells to particle flows [9][10][11]. This system uses vacuum channels on both sides of the central cell channel, enabling the cyclic stretching of the membrane to mimic various breathing scenarios, as depicted in Fig. 1. ...
The"Breathing Lung-on-a-Chip,"a novel microfluidic device featuring a stretchable membrane, replicates the natural expansion and contraction of the human lung. It provides a more realistic in-vitro platform to study respiratory diseases, particle deposition, and drug delivery mechanisms. This device enables investigations into the effects of inhaled nanoparticles (NPs) on lung tissue and supports the development of advanced inhalation therapies. Uniform and optimal concentration delivery of NPs to cultured cells within the chip is critical, particularly as membrane stretching significantly influences particle dynamics. To address this, we developed a 3D numerical model that accurately simulates NP behavior under dynamic conditions, overcoming experimental limitations. The model, validated against experimental data, explores the effects of flow dynamics, particle size, membrane porosity, and stretching frequency/intensity on NP deposition in the air channel and transfer through the porous membrane into the medium channel. The results indicate that increased membrane stretch enhances the sedimentation rate of NPs in the air channel, thereby promoting their transfer to the medium channel, particularly in membranes with initially low porosity. Additionally, excessive stretching frequencies or intensities can introduce reverse flow and stagnation, leading to a longer residence time for NPs and altering their sedimentation patterns. These insights advance our understanding of NP transport in dynamic lung environments, paving the way for more effective applications of lung-on-a-chip technology in toxicological assessments and respiratory therapy innovations.
Graphical Abstract
... In addition, the air channel also allows the introduction of various airborne substances to model inhalation and study the pathogenesis of various lung diseases. 95,96 B. Cellular models ...
To address the growing need for accurate lung models, particularly in light of respiratory diseases, lung cancer, and the COVID-19 pandemic, lung-on-a-chip technology is emerging as a powerful alternative. Lung-on-a-chip devices utilize microfluidics to create three-dimensional models that closely mimic key physiological features of the human lung, such as the air–liquid interface, mechanical forces associated with respiration, and fluid dynamics. This review provides a comprehensive overview of the fundamental components of lung-on-a-chip systems, the diverse fabrication methods used to construct these complex models, and a summary of their wide range of applications in disease modeling and aerosol deposition studies. Despite existing challenges, lung-on-a-chip models hold immense potential for advancing personalized medicine, drug development, and disease prevention, offering a transformative approach to respiratory health research.
... Organ-on-a-chip is an advanced type of biochip designed to simulate the functions and physiological environments of human organs [1][2][3][4][5][6][7][8][9][10][11]. It has potential applications in precision medicine, toxicological research, and other areas, significantly contributing to the reduction of animal testing. ...
The development of bionic organ-on-a-chip technology relies heavily on advancements in in situ sensors and biochip packaging. By integrating precise biological and fluid condition sensing with microfluidics and electronic components, long-term dynamic closed-loop culture systems can be achieved. This study aims to develop biocompatible heterogeneous packaging and laser surface modification techniques to enable the encapsulation of electronic components while minimizing their impact on fluid dynamics. Using a kidney-on-a-chip as a case study, a non-toxic packaging process and fluid interface control methods have been successfully developed. Experimentally, miniature pressure sensors and control circuit boards were encapsulated using parylene-C, a biocompatible material, to isolate biochemical fluids from electronic components. Ultraviolet laser processing was employed to fabricate structures on parylene-C. The results demonstrate that through precise control of processing parameters, the wettability of the material can be tuned freely within a contact angle range of 60° to 110°. Morphological observations and MTT assays confirmed that the material and the processing methods do not induce cytotoxicity. This technology will facilitate the packaging of various miniature electronic components and biochips in the future. Furthermore, laser processing enables rapid and precise control of interface conditions across different regions within the chip, demonstrating a high potential for customized mass production of biochips. The proposed innovations provide a solution for in situ sensing in organ-on-a-chip systems and advanced biochip packaging. We believe that the development of this technology is a critical step toward realizing the concept of “organ twin”.
... These include computational approaches (Kleinstreuer et al., 2021) and lung-on-a-chip developments, (e.g. Lin et al., 2022), but the greatest focus has been on in vitro cell culture approaches (Singh et al., 2021). Whilst replacing in vivo experiments with other approaches is complex, it generally has the advantage that human cell cultures are used, thus reducing issues with interspecies variations. ...
... In another model, paraquat poisoning was used to induce pulmonary fibrosis in a chip with an epithelial and interstitial interface [206]. For inhaled toxins, models incorporating methods for direct gas/aerosol exposure to vascularized airway cells have been developed [207][208][209]. Pathological markers induced by inhaled toxins, indicating conditions such as asthma and COPD, can then be analyzed with the chip system [210]. ...
... However, the current drug evaluation approaches such as animal model experiments, have some limitations in terms of high cost, the existence of species differences, and ethical issues. 8 Moreover, traditional two-dimensional (2D) cell culture techniques lack the complexity of human physiological systems, leading to misleading of results drug screening occasionally. 9 To address these challenges, scientists have been exploring more efficient methods, such as the development of three-dimensional (3D) cultured tissue models in vitro. ...
Cardiovascular disease (CVD) is currently a serious and growing public health problem. In tackling this challenge, organ‐on‐chip (OoC) technology, combined with cell culture and microfluidics, presents a powerful approach for constructing sophisticated tissue models in vitro that can simulate the physiological and pathological microenvironments of human organs. Nowadays, OoC technology has emerged as a pivotal tool in advancing our understanding of CVD pathogenesis, facilitating tissue regeneration studies, conducting efficient drug screening, and assessing therapeutic effects. Moreover, it offers a diverse array of study platforms for preclinical research, fostering innovative approaches towards combating CVD and improving patient outcomes. In this review, we first present the key advantages of OoC technology, including its highly relevant physiological microenvironment, incorporation of integrated functions, and the possibility of the construction of multiorgan‐on‐a‐chip through microfluidic linkage. Then, we summarized the role of OoC in the construction of disease pathological models, which provides a new channel for the exploration of disease pathological mechanisms. Moreover, we discuss the application of this technology in cardiac regeneration and drug screening. Finally, we discuss the challenges of tissue models constructed based on OoC technology and the prospects of this innovative approach.
... The OoC models provide compelling evidence for the use of in vitro human lung models in disease modeling, drug discovery, and drug testing, surpassing the limitations of animal models in accurately simulating the structure, disease symptoms, and responses of the human respiratory system (Lin et al., 2022). ...
Animal testing is required in drug development research and is crucial for assessing the efficacy and safety of medications before they are commercialized. However, the newly furnished Food and Drug Administration Modernization Act 2.0 has given new insight into drug development. It opens a new door by offering an alternative testing method for developing a new drug without using animals. This newly proposed system may potentially significantly impact nondeveloped countries worldwide. In this study, we explore the alternative testing options such as in silico modeling, human tissue-on-chip engineering, animal-free recombinant antibodies, tissue engineering, and artificial intelligence presented by this act and discuss its implications for nondeveloped countries.
... OOCs are robust platforms with a wide range of applications in medical engineering, which generally include the four main components: microfluidic chips, living cells, components for cell stimulation and maturation, and sensors for reading the results [25]. These systems were developed for some organs, such as the liver [26], heart, lung [27], and on coimmunology [28], that play an essential role in drug screening, disease modeling, and personalized medicine [13,24]. OOCs are microfluidic devices for culturing living cells in microchambers that aim to build at least one functional unit of tissue to model its physiological functions. ...
Cardiovascular diseases are caused by hereditary factors, environmental conditions, and medication-related issues. On the other hand, the cardiotoxicity of drugs should be thoroughly examined before entering the market. In this regard, heart-on-chip (HOC) systems have been developed as a more efficient and cost-effective solution than traditional methods, such as 2D cell culture and animal models. HOCs must replicate the biology, physiology, and pathology of human heart tissue to be considered a reliable platform for heart disease modeling and drug testing. Therefore, many efforts have been made to find the best methods to fabricate different parts of HOCs and to improve the bio-mimicry of the systems in the last decade. Beating HOCs with different platforms have been developed and techniques, such as fabricating pumpless HOCs, have been used to make HOCs more user-friendly systems. Recent HOC platforms have the ability to simultaneously induce and record electrophysiological stimuli. Additionally, systems including both heart and cancer tissue have been developed to investigate tissue-tissue interactions' effect on cardiac tissue response to cancer drugs. In this review, all steps needed to be considered to fabricate a HOC were introduced, including the choice of cellular resources, biomaterials, fabrication techniques, biomarkers, and corresponding biosensors. Moreover, the current HOCs used for modeling cardiac diseases and testing the drugs are discussed. We finally introduced some suggestions for fabricating relatively more user-friendly HOCs and facilitating the commercialization process.
... Epi, endo The epithelial stimulation with inflammatory cytokine tissue necrosis factor alpha [221,222] Epi (line) The toxicity due to the silica nanoparticles in the pulmonary region [198,223] Epi, endo Pulmonary oedema caused via the interleukin-2 which is detected by the leakage of fluids [120,224] Epi, endo The infiltration from the neutrophils [225,226] Epi (line), endo, immune Virus infection (SARS-CoV-2), inflammation [227] Epi, endo, cancer Lung cancer [212] Epi COPD induced by smoke [228] Epi (line), endo, immune Asthma, COPD [191,229] Epi Mechanical injury to airway cells [230] Epi (line), endo, immune, bacteria Cystic fibrosis, inflammation, bacterial infection [231] Epi, endo, immune Virus infection (influenza, pseudotyped SARS-CoV-2), inflammation [232] Skin Keratinocyte Model for wound healing, inflammation, repair, irritation, ageing and shear stress studies [233] Gut Epi (org), endo Intestinal differentiation [145] Epi (line), endo The epithelium infected by virus coxsackie B1 The pathogen-induced injury [234] Epi (line), endo The peristaltic mechanical deformation-induced bacterial out-growth [235,236] Epi (line), bacteria Host-microbiome interactions (to clinically simulate effects of microbiome metabolites on host) [237] Epi (line) The loss of the barrier function caused by staurosporine and aspirin [238] Epi (line), endo, lymphatic, endo, immune, bacteria Bacterial infection and inflammation [144] Epi (line) Enteric virus infection (to clinically mimic infection-associated injury) [239] Gut Epi (org), immune Inflammatory bowel disease (IBD) [240] Epi (org), endo, immune, virus Enteric virus infection [241] Epi (line), endo, virus SARS-CoV-2 virus infection [242] Epi (org), bacteria Bacterial infection, mechano-sensitivity (to clinically mimic Shigella infection) [243] Epi (line), endo, Radiation injury [244] Kidney ...
Over the decades, conventional in vitro culture systems and animal models have been used to study physiology, nutrient or drug metabolisms including mechanical and physiopathological aspects. However, there is an urgent need for Integrated Testing Strategies (ITS) and more sophisticated platforms and devices to approach the real complexity of human physiology and provide reliable extrapolations for clinical investigations and personalized medicine. Organ-on-a-chip (OOC), also known as a microphysiological system, is a state-of-the-art microfluidic cell culture technology that sums up cells or tissue-to-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology, and it has been developed to fill the gap between in vitro experimental models and human pathophysiology. The wide range of OOC platforms involves the miniaturization of cell culture systems and enables a variety of novel experimental techniques. These range from modeling the independent effects of biophysical forces on cells to screening novel drugs in multi-organ microphysiological systems, all within microscale devices. As in living biosystems, the development of vascular structure is the salient feature common to almost all organ-on-a-chip platforms. Herein, we provide a snapshot of this fast-evolving sophisticated technology. We will review cutting-edge developments and advances in the OOC realm, discussing current applications in the biomedical field with a detailed description of how this technology has enabled the reconstruction of complex multi-scale and multifunctional matrices and platforms (at the cellular and tissular levels) leading to an acute understanding of the physiopathological features of human ailments and infections in vitro.
... Most of the cell culture approaches involve monolayer cultures of lung primary cells or established cell lines (e.g., A549, BEAS-2B), although more sophisticated cell cultures, such as three-dimensional spheroids [9], air-liquid interface models (ALI) [10] or lung-on-a-chip methods [11], are being progressively introduced to enhance the physiological relevance of the toxicological results. ...
Air pollution constitutes an environmental problem that it is known to cause many serious adverse effects on the cardiovascular and respiratory systems. The chemical characterization of particulate matter (PM) is key for a better understanding of the associations between chemistry and toxicological effects. In this work, the chemical composition and biological effects of fifteen PM10 air filter samples from three air quality stations in Catalonia with contrasting air quality backgrounds were investigated. Three-dimensional (3D) lung cancer cell cultures were exposed to these sample extracts, and cytotoxicity, reactive oxygen species (ROS) induction, metabolomics, and lipidomics were explored. The factor analysis method Multivariate Curve Resolution–Alternating Least-Squares (MCR-ALS) was employed for an integrated interpretation of the associations between chemical composition and biological effects, which could be related to urban traffic emission, biomass burning smoke, and secondary aerosols. In this pilot study, a novel strategy combining new approach methodologies and chemometrics provided new insights into the biomolecular changes in lung cells associated with different sources of air pollution. This approach can be applied in further research on air pollution toxicity to improve our understanding of the causality between chemistry and its effects.
The development of a inhaled nanodrug delivery assessment platform is crucial for advancing treatments for chronic lung diseases. Traditional in vitro models and commercial aerosol systems fail to accurately simulate the complex human respiratory patterns and mucosal barriers. To address this, we have developed the breathing mucociliary-on-a-chip (BMC) platform, which replicates mucociliary clearance and respiratory dynamics in vitro. This platform allows for precise analysis of drug deposition and penetration, providing critical insights into how liposomes and other nanocarriers interact with lung tissues under various airflow conditions. Our results reveal that liposomes penetrate deeper into the cellular layer under high shear stress, with both static and dynamic airflows distinctly affecting their drug release rates. The BMC platform integrates dynamic inhalation systems with mucociliary clearance functionality, enabling a comprehensive evaluation of drug delivery efficacy. This approach highlights the importance of airflow dynamics in optimizing inhalable nanodrug delivery systems, improving nanocarrier design, and tailoring drug dosages and release strategies. The BMC platform represents a significant advancement in the field of inhaled nanodrug delivery, offering a more accurate and reliable method for assessing the performance of therapies. By providing a detailed understanding of drug interactions with lung tissues, this platform supports the development of personalized inhaled therapies and offers promising strategies for treating pulmonary diseases and advancing nanodrug development.
Current in-vitro 2D cultures and animal models present severe limitations in recapitulating human physiopathology with striking discrepancies in estimating drug efficacy and side effects when compared to human trials. For these reasons, microphysiological systems, organ-on-chip and multiorgans microdevices attracted considerable attention as novel tools for high-throughput and high-content research to achieve an improved understanding of diseases and to accelerate the drug development process towards more precise and eventually personalized standards. This review takes the form of a guide on this fast-growing field, providing useful introduction to major themes and indications for further readings. We start analyzing Organs-on-chips (OOC) technologies for testing the major drug administration routes: (1) oral/rectal route by intestine-on-a-chip, (2) inhalation by lung-on-a-chip, (3) transdermal by skin-on-a-chip and (4) intravenous through vascularization models, considering how drugs penetrate in the bloodstream and are conveyed to their targets. Then, we focus on OOC models for (other) specific organs and diseases: (1) neurodegenerative diseases with brain models and blood brain barriers, (2) tumor models including their vascularization, organoids/spheroids, engineering and screening of antitumor drugs, (3) liver/kidney on chips and multiorgan models for gastrointestinal diseases and metabolic assessment of drugs and (4) biomechanical systems recapitulating heart, muscles and bones structures and related diseases. Successively, we discuss technologies and materials for organ on chips, analyzing (1) microfluidic tools for organs-on-chips, (2) sensor integration for real-time monitoring, (3) materials and (4) cell lines for organs on chips. (Nano)delivery approaches for therapeutics and their on chip assessment are also described. Finally, we conclude with a critical discussion on current significance/relevance, trends, limitations, challenges and future prospects in terms of revolutionary impact on biomedical research, preclinical models and drug development.
Conventional preclinical evaluation implemented in 2D monolayer cultures falls short with multiorgan interactions, while animal systems lack the specificity of the human species. Thus, failure in drug efficacy in clinical trials is the paramount hurdle faced by pharmaceutical ventures. Moreover, most of the analysis is based on off-chip and imaging technology. Compared to conventional technology, biosensors have provided sensitive, selective, on-site, and real-time monitoring of microbiophysiological signals for diagnostics and therapeutic applications related to tissue engineering. In addition, a new class of advanced microfluidic-based organ-on-a-chip (OoC) and multiple organ-on-a-chip (MOoC) systems have been trending significantly in the development of drug studies due to their patient-specific evaluation of drug effects. The OoC systems, also referred to as microphysiological systems, have the precise ability to replicate the key features of the cell environment in its native form and thus can mimic human organs. Further, in MOoC systems, several individual OoC devices are held together in a physiological pattern to overcome the structural and functional integrity in an in vitro human model. In this review, we briefly demonstrate biosensor-integrated OoC and MOoC platforms, which will be game-changers in the restructuring of clinical trials. We broadly cover heart-on-a-chip, blood–brain barrier-on-a chip, lung-on-a-chip, liver-on-a chip, kidney-on-a chip, pancreas-on-a-chip, and human-on-a-chip systems integrated with various optical, physical, and electrochemical biosensors. Furthermore, we elaborate combining technologies with potential innovative function of biosensors for the prediction, evaluation, discovery, diagnostics, and therapeutics of human ailments. Finally, the limitations and future prospects are discussed from an end-user perspective.
The microfluidic and lab-on-a-chip (LoC) devices are promising for disease modeling and drug screening. New bio-imaging agents that permit multiple imaging and therapeutic applications can lead to more accurate diagnosis and a better understanding of local diseased tissue. The spectroscopic properties of nanomaterials, such as carbon dots, can be continuously monitored using an external capillary flow, which allows efficient monitoring over fluorescence peaks and size-dependent synthesis. The microfluidic devices also offer a relatively simple and consistent way of overcoming issues that come with conventional methods of nanoparticle fabrication for drug delivery and controlled release. The microfluidic devices have been used to generate encapsulated cells in droplets or microgels, thereby providing new opportunities for fundamental studies in cell behavior, gene expression, and enzymatic activity. Several materials, including collagen, gelatin, fibrin, polypeptides, alginate, hyaluronic acid, chitosan, agarose, poly(ethylene glycol), and poly(ethylene glycol) diacrylate, have been widely used to develop microfluidic devices for different applications. The encapsulation of cells within polymer particles can be achieved by adjusting the flow rate between the continuous and dispersed phases, and using a high aspect-ratio microchannel. Most toxicological testing is conducted on animal models following the regulatory testing requirements. The animal models have provided considerable experimental results and insights into lung physiology and pathophysiology. However, due to the structural limitations, disease symptoms, and responses of the human respiratory system, a sophisticated in vitro model is required to analyze the drug efficacy, toxicity, and different diseases. This chapter provides concise information on the progress of microfluidics and LoC-based devices and their potential biomedical applications.
The lungs are affected by illnesses including asthma, chronic obstructive pulmonary disease, and infections such as influenza and SARS-CoV-2. Physiologically relevant models for respiratory conditions will be essential for new drug development. The composition and structure of the lung extracellular matrix (ECM) plays a major role in the function of the lung tissue and cells. Lung-on-chip models have been developed to address some of the limitations of current two-dimensional in vitro models. In this review, we describe various ECM substitutes utilized for modeling the respiratory system. We explore the application of lung-on-chip models to the study of cigarette smoke and electronic cigarette vapor. We discuss the challenges and opportunities related to model characterization with an emphasis on in situ characterization methods, both established and emerging. We discuss how further advancements in the field, through the incorporation of interstitial cells and ECM, have the potential to provide an effective tool for interrogating lung biology and disease, especially the mechanisms that involve the interstitial elements.
Faster, cheaper, sensitive, and mechanisms‐based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM‐induced lung fibrosis, are proposed.
The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks. Zamprogno et al. report a lung-on-a-chip of the second generation that mimics an array of alveoli with in vivo-like dimensions, based on a thin, stretchable collagen-elastin membrane used to reproduce the air-blood barrier functions. The membrane is stable, can be cultured on both sides for weeks, is biodegradable and its elastic properties allow mimicking respiratory motions by mechanically stretching the cells.
Multiorgan-on-a-chip (multi-OoC) platforms have great potential to redefine the way in which human health research is conducted. After briefly reviewing the need for comprehensive multiorgan models with a systemic dimension, we highlight scenarios in which multiorgan models are advantageous. We next overview existing multi-OoC platforms, including integrated body-on-a-chip devices and modular approaches involving interconnected organ-specific modules. We highlight how multi-OoC models can provide unique information that is not accessible using single-OoC models. Finally, we discuss remaining challenges for the realization of multi-OoC platforms and their worldwide adoption. We anticipate that multi-OoC technology will metamorphose research in biology and medicine by providing holistic and personalized models for understanding and treating multisystem diseases.
Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus–host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air–liquid interface, organoids, or ‘on-chip’ technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium—the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.
The novel coronavirus COVID-19 appears to strike some people more intensely than others. Some people only experience mild symptoms while others require hospitalization and ventilation. With the virus becoming more prevalent day by day, it is not just the elderly, but even young people are falling seriously ill. Various researchers across the world state that specific cells in the nasal passages, intestines, and lungs may be more susceptible to the infection. Shifting the focus and research towards epithelium might provide new insight towards understanding COVID-19. This article is an overview of how epithelium permeability in COVID-19 may associate with comorbidities and other factors.
When assessing the risk and hazard of a non-pharmaceutical compound, the first step is determining acute toxicity, including toxicity following inhalation. Inhalation is a major exposure route for humans, and the respiratory epithelium is the first tissue that inhaled substances directly interact with. Acute inhalation toxicity testing for regulatory purposes is currently performed only in rats and/or mice according to OECD TG403, TG436, and TG433 test guidelines. Such tests are biased by the differences in the respiratory tract architecture and function across species, making it difficult to draw conclusions on the potential hazard of inhaled compounds in humans. Research efforts have been therefore focused on developing alternative, human-relevant models, with emphasis on the creation of advanced In vitro models. To date, there is no In vitro model that has been accepted by regulatory agencies as a stand-alone replacement for inhalation toxicity testing in animals. Here, we provide a brief introduction to current OECD test guidelines for acute inhalation toxicity, the interspecies differences affecting the predictive value of such tests, and the current regulatory efforts to advance alternative approaches to animal-based inhalation toxicity studies. We then list the steps that should allow overcoming the current challenges in validating In vitro alternatives for the successful replacement of animal-based inhalation toxicity studies. These steps are inclusive and descriptive, and should be detailed when adopting in house-produced 3D cell models for inhalation tests. Hence, we provide a checklist of key parameters that should be reported in any future scientific publications for reproducibility and transparency.
Air pollution consists of highly variable and complex mixtures recognized as major contributors to morbidity and mortality worldwide. The vast number of chemicals, coupled with limitations surrounding epidemiological and animal studies, has necessitated the development of new approach methods (NAMs) to evaluate air pollution toxicity. These alternative approaches include in vitro (cell-based) models, wherein toxicity of test atmospheres can be evaluated with increased efficiency compared to in vivo studies. In vitro exposure systems have recently been developed with the goal of evaluating air pollutant-induced toxicity; though the specific design parameters implemented in these NAMs-based studies remain in flux. This review aims to outline important design parameters to consider when using in vitro methods to evaluate air pollutant toxicity, with the goal of providing increased accuracy, reproducibility, and effectiveness when incorporating in vitro data into human health evaluations. This review is unique in that experimental considerations and lessons learned are provided, as gathered from first-hand experience developing and testing in vitro models coupled to exposure systems. Reviewed design aspects include cell models, cell exposure conditions, exposure chambers, and toxicity endpoints. Strategies are also discussed to incorporate in vitro findings into the context of in vivo toxicity and overall risk assessment.
Endothelial cells (ECs) are continuously subjected to fluid wall shear stress (WSS) and cyclic strain caused by pulsatile blood flow and pressure. It is well established that these hemodynamic forces each play important roles in vascular disease, but their combined effects are not well understood. ECs remodel in response to both WSS and cyclic strain to align along the vessel axis, but in areas prone to atherogenesis, such an alignment is absent. In this perspective, experimental and clinical findings will be reviewed, which have revealed the characteristics of WSS and cyclic strain, which are associated with atherosclerosis, spanning studies on whole blood vessels to individual cells to mechanosensing molecules. Examples are described regarding the use of computational modeling to elucidate the mechanisms by which EC alignment contributes to mechanical homeostasis. Finally, the need to move toward an integrated understanding of how hemodynamic forces influence EC mechanotransduction is presented, which holds the potential to move our currently fragmented understanding to a true appreciation of the role of mechanical stimuli in atherosclerosis.
In multi-ciliated cells, directed and synchronous ciliary beating in the apical membrane occurs through appropriate configuration of basal bodies (BBs, roots of cilia). Although it has been experimentally shown that the position and orientation of BBs are coordinated by apical cytoskeletons (CSKs), such as microtubules (MTs), and planar cell polarity (PCP), the underlying mechanism for achieving the patterning of BBs is not yet understood. In this study, we propose that polarity in bundles of apical MTs play a crucial role in the patterning of BBs. First, the necessity of the polarity was discussed by theoretical consideration on the symmetry of the system. The existence of the polarity was investigated by measuring relative angles between the MTs and BBs using published experimental data. Next, a mathematical model for BB patterning was derived by combining the polarity and self-organizational ability of CSKs. In the model, BBs were treated as finite-size particles in the medium of CSKs and excluded volume effects between BBs and CSKs were taken into account. The model reproduces the various experimental observations, including normal and drug-treated phenotypes. Our model with polarity provides a coherent and testable mechanism for apical BB pattern formation. We have also discussed the implication of our study on cell chirality.
The organ-on-a-chip (OOAC) is in the list of top 10 emerging technologies and refers to a physiological organ biomimetic system built on a microfluidic chip. Through a combination of cell biology, engineering, and biomaterial technology, the microenvironment of the chip simulates that of the organ in terms of tissue interfaces and mechanical stimulation. This reflects the structural and functional characteristics of human tissue and can predict response to an array of stimuli including drug responses and environmental effects. OOAC has broad applications in precision medicine and biological defense strategies. Here, we introduce the concepts of OOAC and review its application to the construction of physiological models, drug development, and toxicology from the perspective of different organs. We further discuss existing challenges and provide future perspectives for its application.
Mucociliary clearance (MCC) is one of the most important defence mechanisms of the human respiratory system. Its failure is implicated in many chronic and debilitating airway diseases. However, due to the complexity of lung organization, we currently lack full understanding on the relationship between these regional differences in anatomy and biology and MCC functioning. For example, it is unknown whether the regional variability of airway geometry, cell biology and ciliary mechanics play a functional role in MCC. It therefore remains unclear whether the regional preference seen in some airway diseases could originate from local MCC dysfunction. Though great insights have been gained into the genetic basis of cilia ultrastructural defects in airway ciliopathies, the scaling to regional MCC function and subsequent clinical phenotype remains unpredictable. Understanding the multiscale mechanics of MCC would help elucidate genotype–phenotype relationships and enable better diagnostic tools and treatment options. Here, we review the hierarchical and variable organization of ciliated airway epithelium in human lungs and discuss how this organization relates to MCC function. We then discuss the relevancy of these structure–function relationships to current topics in lung disease research. Finally, we examine how state-of-the-art computational approaches can help address existing open questions.
This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.
Advances in the biological sciences have led to an ongoing paradigm shift in toxicity testing based on expanded application of high-throughput in vitro screening and in silico methods to assess potential health risks of environmental agents. This review examines progress on the vision for toxicity testing elaborated by the US National Research Council (NRC) during the decade that has passed since the 2007 NRC report on Toxicity Testing in the 21st Century (TT21C). Concomitant advances in exposure assessment, including computational approaches and high-throughput exposomics, are also documented. A vision for the next generation of risk science, incorporating risk assessment methodologies suitable for the analysis of new toxicological and exposure data, resulting in human exposure guidelines is described. Case study prototypes indicating how these new approaches to toxicity testing, exposure measurement, and risk assessment are beginning to be applied in practice are presented. Overall, progress on the 20-year transition plan laid out by the US NRC in 2007 has been substantial. Importantly, government agencies within the United States and internationally are beginning to incorporate the new approach methodologies envisaged in the original TT21C vision into regulatory practice. Future perspectives on the continued evolution of toxicity testing to strengthen regulatory risk assessment are provided.
Bacterial invasion of the respiratory system leads to complex immune responses. In the deep alveolar regions, the first line of defense includes foremost the alveolar epithelium, the surfactant‐rich liquid lining, and alveolar macrophages. Typical in vitro models come short of mimicking the complexity of the airway environment in the onset of airway infection; among others, they neither capture the relevant anatomical features nor the physiological flows innate of the acinar milieu. Here, novel microfluidic‐based acini‐on‐chips that mimic more closely the native acinar airways at a true scale with an anatomically inspired, multigeneration alveolated tree are presented and an inhalation‐like maneuver is delivered. Composed of human alveolar epithelial lentivirus immortalized cells and macrophages‐like human THP‐1 cells at an air–liquid interface, the models maintain critically an epithelial barrier with immune function. To demonstrate, the usability and versatility of the platforms, a realistic inhalation exposure assay mimicking bacterial infection is recapitulated, whereby the alveolar epithelium is exposed to lipopolysaccharides droplets directly aerosolized and the innate immune response is assessed by monitoring the secretion of IL8 cytokines. These efforts underscore the potential to deliver advanced in vitro biosystems that can provide new insights into drug screening as well as acute and subacute toxicity assays.
Organs and tissues in multi-cellular organisms exhibit various morphologies. Tubular organs have multi-scale morphological features which are closely related to their functions. Here we discuss morphogenesis and the mechanical functions of the vertebrate oviduct in the female reproductive tract, also known as the fallopian tube. The oviduct functions to convey eggs from the ovary to the uterus. In the luminal side of the oviduct, the epithelium forms multiple folds (or ridges) well-aligned along the longitudinal direction of the tube. In the epithelial cells, cilia are formed orienting toward the downstream of the oviduct. The cilia and the folds are supposed to be involved in egg transportation. Planar cell polarity (PCP) is developed in the epithelium, and the disruption of the Celsr1 gene, a PCP related-gene, causes randomization of both cilia and fold orientations, discontinuity of the tube, inefficient egg transportation, and infertility. In this review article, we briefly introduce various biophysical and biomechanical issues in the oviduct, including physical mechanisms of formation of PCP and organized cilia orientation, epithelial cell shape regulation, fold pattern formation generated by mechanical buckling, tubulogenesis, and egg transportation regulated by fluid flow. We also mention about possible roles of the oviducts in egg shape formation and embryogenesis, sinuous patterns of tubes, and fold and tube patterns observed in other tubular organs such as the gut, airways, etc.
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Background:
Millions of workers are exposed to carcinogenic polycyclic aromatic hydrocarbon (PAH) mixtures. The toxicity of PAH mixtures is variable and depends on the composition of the mixture, which is related to the emission sources. Although several indicators exist, the cancer risk estimation associated with occupational exposure to PAHs is poorly known.
Objectives:
To assess the risk of lung cancer associated with PAHs in several industries using the atmospheric concentrations of benzo[a]pyrene (BaP) as a proxy.
Methods:
A total of 93 exposure groups belonging to 9 industries were investigated. Eight indicators found in the literature were compared to assess risks. A consensual indicator was used to estimate lung cancer risks.
Results:
Approximately 30% of the exposure groups were above the maximal risk level of the European Union (10-4). The risk probabilities were >10-3 for coke and silicon production; >10-4 for the manufacturing of carbon products and aluminum production; >10-5 for foundries and combustion processes; >10-6 for the use of lubricating oils and engine exhaust emissions; and >10-7 for bitumen. The risk probabilities were highly variable within industries (from 1 to 1000 likelihood). A total of 27 (95% CI: 0.1-54) contemporary additional lung cancer cases could be expected per year in the French exposed population based on estimations using published data.
Conclusion:
This study provides an overview of cancer risk estimation in many industries. Despite efforts and changes that had been made to decrease risks, PAHs remain a sanitary threat for people exposed to these pollutants in occupational environments.
Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.
Recent studies have revealed biologically significant differences between human and mouse lung development, and have reported new in vitro systems that allow experimental manipulation of human lung models. At the same time, emerging clinical data suggest that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. In this Review, we discuss our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments using in vitro human lung developmental models and RNA sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on the mechanisms underlying human lung development and disease, and may help pave the way for new therapies.
Introduction:
Knowledge of acute inhalation toxicity potential is important for establishing safe use of chemicals and consumer products. Inhalation toxicity testing and classification procedures currently accepted within worldwide government regulatory systems rely primarily on tests conducted in animals. The goal of the current work was to develop and prevalidate a nonanimal (in vitro) test for determining acute inhalation toxicity using the EpiAirway™ in vitro human airway model as a potential alternative for currently accepted animal tests.
Materials and Methods:
The in vitro test method exposes EpiAirway tissues to test chemicals for 3 hours, followed by measurement of tissue viability as the test endpoint. Fifty-nine chemicals covering a broad range of toxicity classes, chemical structures, and physical properties were evaluated. The in vitro toxicity data were utilized to establish a prediction model to classify the chemicals into categories corresponding to the currently accepted Globally Harmonized System (GHS) and the Environmental Protection Agency (EPA) system.
Results:
The EpiAirway prediction model identified in vivo rat-based GHS Acute Inhalation Toxicity Category 1-2 and EPA Acute Inhalation Toxicity Category I-II chemicals with 100% sensitivity and specificity of 43.1% and 50.0%, for GHS and EPA acute inhalation toxicity systems, respectively. The sensitivity and specificity of the EpiAirway prediction model for identifying GHS specific target organ toxicity-single exposure (STOT-SE) Category 1 human toxicants were 75.0% and 56.5%, respectively. Corrosivity and electrophilic and oxidative reactivity appear to be the predominant mechanisms of toxicity for the most highly toxic chemicals.
Conclusions:
These results indicate that the EpiAirway test is a promising alternative to the currently accepted animal tests for acute inhalation toxicity.
Background:
Planar cell polarity (PCP) coordinates the patterning and orientation of cells and their structures along tissue planes, and although its acquisition during the formation of airway epithelium has been described, the mechanisms for its maintenance and reconstruction are poorly understood. We aimed to clarify whether ambient environment change by orthotropic autologous transplantation affected PCP at the cellular level.
Methods:
We performed orthotropic autologous transplantation by inverting tracheal segments in rats, and then performed morphological evaluation by microscopy. The PCP of the tracheal epithelium was assessed over time by analyzing the directions of mucociliary transport and ciliary beat, the positional relationship between the basal body and basal foot, and the bias of Vang-like protein 1 (Vangl1) at 2, 4, and 6 months postoperatively.
Results:
After 2 months, the directions of mucociliary transport and ciliary beat were preserved toward the lung in the inverted tracheal segments. The positional relationship between the basal body and the basal foot, and the bias of Vangl1, also indicated preservation of PCP in the inverted tracheal segments. Similar results were obtained at 6 months.
Conclusion:
The PCP of ciliated epithelium was preserved in reversed trachea, even after long-term observation.
Inhalation toxicity testing, which provides the basis for hazard labeling and risk management of chemicals with potential exposure to the respiratory tract, has traditionally been conducted using animals. Significant research efforts have been directed at the development of mechanistically based, non-animal testing approaches that hold promise to provide human-relevant data and an enhanced understanding of toxicity mechanisms. A September 2016 workshop, "Alternative Approaches for Acute Inhalation Toxicity Testing to Address Global Regulatory and Non-Regulatory Data Requirements", explored current testing requirements and ongoing efforts to achieve global regulatory acceptance for non-animal testing approaches. The importance of using integrated approaches that combine existing data with in vitro and/or computational approaches to generate new data was discussed. Approaches were also proposed to develop a strategy for identifying and overcoming obstacles to replacing animal tests. Attendees noted the importance of dosimetry considerations and of understanding mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Recommendations were made to (1) develop a database of existing acute inhalation toxicity data; (2) prepare a state-of-the-science review of dosimetry determinants, mechanisms of toxicity, and existing approaches to assess acute inhalation toxicity; (3) identify and optimize in silico models; and (4) develop a decision tree/testing strategy, considering physicochemical properties and dosimetry, and conduct proof-of-concept testing. Working groups have been established to implement these recommendations.
Bronchial asthma is characterized by persistent cough, increased sputum, and repeated wheezing. The pathophysiology underlying these symptoms is the hyper-responsiveness of the airway along with chronic airway inflammation. Repeated injury, repair, and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium; such changes are believed to have a significant association with the pathophysiology of asthma. Damage to the barrier functions of the airway epithelium enhances mucosal permeability of foreign substances in the airway epithelium of patients with asthma. Thus, epithelial barrier fragility is closely involved in releasing epithelial cytokines (e.g., TSLP, IL-25, and IL-33) because of the activation of airway epithelial cells, dendritic cells, and innate group 2 innate lymphoid cells (ILC2). Functional abnormalities of the airway epithelial cells along with the activation of dendritic cells, Th2 cells, and ILC2 form a single immunopathological unit that is considered to cause allergic airway inflammation. Here we use the latest published literature to discuss the potential pathological mechanisms regarding the onset and progressive severity of asthma with regard to the disruption of the airway epithelial function.
To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.
Pulmonary thrombosis is a significant cause of patient mortality, however, there are no effective in vitro models of thrombi formation in human lung microvessels, that could also assess therapeutics and toxicology of antithrombotic drugs. Here we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium, and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development. This article is protected by copyright. All rights reserved.
The REACH Regulation requires information on acute oral toxicity for substances produced or imported in quantities greater than one tonne per year. When registering, animal testing should be used as last resort. The standard acute oral toxicity test requires use of animals. Therefore, the European Chemicals Agency examined whether alternative ways exist to generate information on acute oral toxicity. The starting hypothesis was that low acute oral toxicity can be predicted from the results of low toxicity in oral sub-acute toxicity studies. Proving this hypothesis would allow avoiding acute toxicity oral testing whenever a sub-acute oral toxicity study is required or available and indicates low toxicity. ECHA conducted an analysis of the REACH database and found suitable studies on both acute oral and sub-acute oral toxicities for 1,256 substances. 415 of these substances had low toxicity in the sub-acute toxicity study (i.e. NO(A)EL at or above the classification threshold of 1,000 mg/kg). For 98% of these substances, low acute oral toxicity was also reported (i.e. LD₅₀ above the classification threshold of 2,000 mg/kg). On the other hand, no correlation was found between lower NO(A)ELs and LD₅₀. According to the REACH regulation, this approach for predicting acute oral toxicity needs to be considered as part of a weight of evidence analysis. Therefore, additional sources of information to support this approach are presented. Ahead of the last REACH registration deadline in 2018, ECHA estimates that registrants of about 550 substances can omit the in vivo acute oral study by using this adaptation.
Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.
Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multicili-ated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.
Recently, a continuous increase in environmental pollution has been observed. Despite wide-scale efforts to reduce air pollutant emissions, the problem is still relevant. Exposure to elevated levels of airborne particles increased the incidence of respiratory diseases. PM10 constitute the largest fraction of air pollutants, containing particles with a diameter of less than 10 μm, metals, pollens, mineral dust and remnant material from anthropogenic activity. The natural airway defensive mechanisms against inhaled material, such as mucus layer, ciliary clearance and macrophage phagocytic activity, may be insufficient for proper respiratory function. The epithelium layer can be disrupted by ongoing oxidative stress and inflammatory processes induced by exposure to large amounts of inhaled particles as well as promote the development and exacerbation of obstructive lung diseases. This review draws attention to the current state of knowledge about the physical features of PM10 and its impact on airway epithelial cells, and obstructive pulmonary diseases.
Silica nanoparticles (SiNPs) are one of the most widely used types of nanoparticles across many industrial sectors, and are known to be present in the air year-round. In this study, we aimed to evaluate the potential adverse effects of SiNP exposure on pulmonary epithelial tight junctions, which serve as a critical barrier between the respiratory system and the circulatory system. In vivo studies confirmed that SiNPs decreased the protein expression levels of zonula occludens 1 (ZO-1), zonula occludens 2 (ZO-2), and occludin in the lungs of C57BL/6 mice. In vitro studies showed that SiNPs not only decreased the mRNA and protein expression of ZO-1 and ZO-2, but also decreased the protein expression of occludin in human bronchial epithelial (BEAS-2B) cells. In addition, SiNP exposure increased reactive oxygen species (ROS) production and activated extracellular regulated protein kinases (ERKs) and c-Jun N-terminal kinase (JNK). The inhibition of ROS and ERKs effectively protected the SiNP-induced downregulation of ZO-1 mRNA and protein expression, but had no effect on ZO-2 or occludin expression. SiNP-induced matrix metalloproteinase 9 (MMP9) protein expression appeared to be involved in occludin proteolytic degradation, in addition to SiNP-induced direct occludin protein degradation. The present study suggests that SiNPs disturb pulmonary epithelial tight junction structure and function via the ROS/ERK pathway and protein degradation.
Airborne particulate matters have posed significant risk to human health worldwide. Fine particulate matters (PM2.5, aerodynamic diameter <2.5µm) are associated with increased morbidity and mortality attributed to pulmonary diseases. Advanced in vitro model would benefit the assessment of PM2.5 induced pulmonary injuries and drug development. In this work, we presented a PM2.5 exposure model to evaluate the pulmonary risk of fine particulate matter exposure in an organotypic manner with the help of 3D human lung-on-a-chip. By compartmentalized co-culturing human endothelial cells, epithelial cells and extra cellular matrix, our lung-on-a-chip recapitulated the structural features of the alveolar-blood barrier, which is pivotal for exogenous hazards toxicity evaluation. PM2.5 was applied to the channel lined with lung epithelial cells to model the pulmonary exposure of fine particulate matter. The results indicated acute high dose PM2.5 exposure would lead to various malfunctions of alveolar capillary barrier, including tight junction disruption, increased ROS generation, apoptosis, inflammatory bio-factor expression in epithelial cells and endothelial cells, elevated permeability and monocyte attachments. Collectively, our lung-on-a-chip model provide a simple platform to investigate the complex responses after PM2.5 exposure in a physiological relevant level, which could be of great potential in environmental risk assessment and therapeutic treatment development.
Exposure of lung tissues to cigarette smoke is a major cause of human disease and death worldwide. Unfortunately, adequate model systems that can reliably recapitulate disease biogenesis in vitro, including exposure of the human lung airway to fresh whole cigarette smoke (WCS) under physiological breathing airflow, are lacking. This protocol extension builds upon, and can be used with, our earlier protocol for microfabrication of human organs-on-chips. Here, we describe the engineering, assembly and operation of a microfluidically coupled, multi-compartment platform that bidirectionally ‘breathes’ WCS through microchannels of a human lung small airway microfluidic culture device, mimicking how lung cells may experience smoke in vivo. Several WCS-exposure systems have been developed, but they introduce smoke directly from above the cell cultures, rather than tangentially as naturally occurs in the lung due to lateral airflow. We detail the development of an organ chip–compatible microrespirator and a smoke machine to simulate breathing behavior and smoking topography parameters such as puff time, inter-puff interval and puffs per cigarette. Detailed design files, assembly instructions and control software are provided. This novel platform can be fabricated and assembled in days and can be used repeatedly. Moderate to advanced engineering and programming skills are required to successfully implement this protocol. When coupled with the small airway chip, this protocol can enable prediction of patient-specific biological responses in a matched-comparative manner. We also demonstrate how to adapt the protocol to expose living ciliated airway epithelial cells to smoke generated by electronic cigarettes (e-cigarettes) on-chip. This protocol describes a biomimetic smoking robot that can be used in combination with microfluidic organ chips to simulate disease biogenesis in vitro.
With rapid advances in micro-fabrication processes and the availability of biologically-relevant lung cells, the development of lung-on-chip platforms is offering novel avenues for more realistic inhalation assays in pharmaceutical research, and thereby an opportunity to depart from traditional in vitro lung assays. As advanced models capturing the cellular pulmonary make-up at an air-liquid interface (ALI), lung-on-chips emulate both morphological features and biological functionality of the airway barrier with the ability to integrate respiratory breathing motions and ensuing tissue strains. Such in vitro systems allow importantly to mimic more realistic physiological respiratory flow conditions, with the opportunity to integrate physically-relevant transport determinants of aerosol inhalation therapy, i.e. recapitulating the pathway from airborne flight to deposition on the airway lumen. In this short opinion, we discuss such points and describe how these attributes are paving new avenues for exploring improved drug carrier designs (e.g. shape, size, etc.) and targeting strategies (e.g. conductive vs. respiratory regions) amongst other. We argue that while technical challenges still lie along the way in rendering in vitro lung-on-chip platforms more widespread across the general pharmaceutical research community, significant momentum is steadily underway in accelerating the prospect of establishing these as in vitro "gold standards".
We have developed a pumpless cell culture platform that can recirculate small amounts of cell culture medium (400 µL) in a unidirectional and bidirectional flow pattern. The device produces an average wall shear stress of up to 0.587 Pa ± 0.006 Pa without the use of a pump. It can be used to culture cells that are sensitive to the direction of flow-induced mechanical shear such as human umbilical vein endothelial cells (HUVECs) in a format that allows for large-scale parallel screening of drugs. Using the device we demonstrate that HUVECs produce pro-inflammatory indicators (interleukin 6, interleukin 8) under both flow conditions, but that the secretion was significantly higher under bidirectional flow. Our results show that pumpless devices can simulate the endothelium under healthy and activated conditions depending on the flow pattern chosen. The developed devices and mode of operation can be integrated with pumpless tissues-on-a-chips, allowing for the addition of barrier tissues such as endothelial linings.
A549 cells are common models in the assessment of respiratory cytotoxicity. To provide physiologically more representative exposure conditions and increase the differentiation state, respiratory cells, for instance Calu-3 bronchial epithelial cells, are cultured at an air-liquid interface (ALI). There are indications that A549 cells also change their phenotype upon culture in ALI. The influence of culture in two variations of transwell cultures compared to conventional culture in plastic wells on the phenotype of A549 cells was studied. Cells were characterized by morphology, proliferation and transepithelial electrical resistance, whole genome transcription analysis, Western blot and immunocytochemical detection of pro-surfactant proteins. Furthermore, lipid staining, surface morphology, cell elasticity, surface tension and reaction to quartz particles were performed. Relatively small changes were noted in the expression of differentiation markers for alveolar cells but A549 cells cultured in ALI showed marked differences in lipid staining and surface morphology, surface tension and cytotoxicity of quartz particles. Data show that changes in physiological reactions of A549 cells in ALI culture were rather caused by change of surface properties than by increased expression of surfactant proteins.
Background:
Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings.
Objectives:
The purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell-dependent macrophage activation.
Methods:
Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users.
Results:
The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users.
Conclusions:
Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.
Tumor-infiltrating myeloid cells (TIMs)comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq)to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients’ blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.
Diesel exhaust particles (DEP) are responsible for both respiratory and cardiovascular effects. However many questions are still unravelled and the mechanisms behind the health effects induced by the exposure to ultrafine particles (UFP) need further investigations. Furthermore, different emission sources can lead to diverse biological responses. In this perspective, here we have compared the effects of three DEPs, two standard reference materials (SRM 1650b and 2975) and one DEP directly sampled from a EuroIV vehicle without Diesel Particulate Filter (DPF). For the biological investigations, different in vitro lung models involving both epithelial and vascular endothelial cells, were used. Cell viability, oxidative stress, inflammation, DNA damage and endothelial activation markers were investigated at sub-cytotoxic DEP doses.
The data obtained have shown that only DEP EuroIV, which had the major content of polycyclic aromatic hydrocarbons (PAHs) and metals, was able to induce oxidative stress, inflammation and consequent endothelial activation, as demonstrated by the expression of adhesion molecules (ICAM-1 and VCAM-1) and the release of inflammatory markers (IL-8) from endothelial cells. Standard reference materials were not effective under our experimental conditions. These data suggest that oxidative stress, endothelial activation and systemic inflammatory cytokines release are crucial events after DEP exposure and that the source of DEP emission, responsible of the particle chemical fingerprint, may have a key role in the resulting adverse biological outcomes.
Environmental pollution is one of the largest sources responsible for human diseases and premature death worldwide. However, the methodological development of a spatiotemporally controllable and high-throughput investigation of the environmental pollution-induced biological injury events is still being explored. In this study, we describe a chemical gra-dient generator-aided microfluidic cell system for the dynamic study of representative environmental pollutant-induced bronchial epithelium injury in a throughput manner. We demonstrated the stability and reliability of operation-optimized microfluidic system for precise and long-term chemical gradient production. We also performed a microenvironment-controlled microfluidic bronchial epithelium construction with high viability and structure integration. Moreover, on-chip investigation of bronchial epithelium injury by benzopyrene stimulation with various concentrations can be carried out in the single device. The varying bronchial inflammatory and cytotoxic responses were temporally monitored and measured based on the well-established system. The benzopyrene directionally led the bronchial epithelium to present observable cell shrinkage, cytoskeleton disintegration, Caspase-3 activation, overproduction of reactive oxygen species, and various inflammatory cytokine (TNF-, IL-6, and IL-8) secretion, suggesting its significant inflammatory and cytotoxic effects on respiratory system. We believe the microfluidic advancement has potential applications in the fields of environmental monitoring, tissue engineering, and pharmaceutical development.
The lung is continuously exposed to particles, toxicants, and microbial pathogens that are cleared by a complex mechanical, innate, and acquired immune system. Mucociliary clearance, mediated by the actions of diverse conducting airway and submucosal gland epithelial cells, plays a critical role in a multilayered defense system by secreting fluids, electrolytes, antimicrobial and antiinflammatory proteins, and mucus onto airway surfaces. The mucociliary escalator removes particles and pathogens by the mechanical actions of cilia and cough. Abnormalities in mucociliary clearance, whether related to impaired fluid secretion, ciliary dysfunction, lack of cough, or the disruption of epithelial cells lining the respiratory tract, contribute to the pathogenesis of common chronic pulmonary disorders. Although mucus and other airway epithelial secretions play a critical role in protecting the lung during acute injury, impaired mucus clearance after chronic mucus hyperproduction causes airway obstruction and infection, which contribute to morbidity in common pulmonary disorders, including chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis, and primary ciliary dyskinesia. In this summary, the molecular and cellular mechanisms mediating airway mucociliary clearance, as well as the role of goblet cell metaplasia and mucus hyperproduction, in the pathogenesis of chronic respiratory diseases are considered.
Part of the effective prediction of the pharmacokinetics of drugs (or toxic particles) requires extrapolation of experimental data sets from animal studies to humans. As the respiratory tracts of rodents and humans are anatomically very different, there is a need to study airflow and drug-aerosol deposition patterns in lung airways of these laboratory animals and compare them to those of human lungs. As a first step, interspecies computational comparison modeling of inhaled nano-to-micron size drugs (50 nm < d< 15μm) was performed using mouse and human upper airway models under realistic breathing conditions. Critical species-specific differences in lung physiology of the upper airways and subsequently in local drug deposition were simulated and analyzed. In addition, a hybrid modeling methodology, combining Computational Fluid-Particle Dynamics (CF-PD) simulations with deterministic lung deposition models, was developed and predicted total and regional drug-aerosol depositions in lung airways of both mouse and man were compared, accounting for the geometric, kinematic and dynamic differences. Interestingly, our results indicate that the total particle deposition fractions, especially for submicron particles, are comparable in rodent and human respiratory models for corresponding breathing conditions. However, care must be taken when extrapolating a given dosage as considerable differences were noted in the regional particle deposition pattern. Combined with the deposition model, the particle retention and clearance kinetics of deposited nanoparticles indicates that the clearance rate from the mouse lung is higher than that in the human lung. In summary, the presented computer simulation models provide detailed fluid-particle dynamics results for upper lung airways of representative human and mouse models with a comparative analysis of particle lung deposition data, including a novel mice-to-men correlation as well as a particle-clearance analysis both useful for pharmacokinetic and toxicokinetic studies.
Pathologies of the respiratory system such as lung infections, chronic inflammatory lung diseases, and lung cancer are among the leading causes of morbidity and mortality, killing one in six people worldwide. Development of more effective treatments is hindered by the lack of preclinical models of the human lung that can capture the disease complexity, highly heterogeneous disease phenotypes, and pharmacokinetics and pharmacodynamics observed in patients. The merger of two novel technologies, Organs-on-Chips and human stem cell engineering, has the potential to deliver such urgently needed models. Organs-on-Chips, which are microengineered bioinspired tissue systems, recapitulate the mechanochemical environment and physiological functions of human organs while concurrent advances in generating and differentiating human stem cells promise a renewable supply of patient-specific cells for personalized and precision medicine. Here, we discuss the challenges of modeling human lung pathophysiology in vitro, evaluate past and current models including Organs-on-Chips, review the current status of lung tissue modeling using human pluripotent stem cells, explore in depth how stem-cell based Lung-on-Chips may advance disease modeling and drug testing, and summarize practical consideration for the design of Lung-on-Chips for academic and industry applications.
Determination of acute inhalation toxicity is requested for hazard assessment of substances. In the regulatory-required test, otherwise toxicologically inert solid (dust) aerosols have to be tested up to a very high concentration limit of 5 mg/L of air. By testing a series of organic pigments at this concentration, we found that some pigments were well tolerated (hence, resulting in an LC50 >5 mg/L). For other pigments, we identified obstruction of the airways with subsequent suffocation as the cause of death at this concentration. In these cases, Organization for Economic Cooperation and Development TG 436 requires that additional animals are tested at 1 mg/L air. However, the mortality of animals at the high concentration could have been avoided if the suffocation by obstruction of the airways was predictable. Hence, we investigated the correlation of several physicochemical characteristics with the observed mortality. Test substances with the highest contact angle, a measure of hydrophobicity, produced mortality at high concentrations, whereas the more hydrophilic compounds did not. Therefore, the contact angle of test substances may serve as a predictive parameter for suffocation potential. We propose conducting this characterization before in vivo testing to reduce the number and suffering of animals until further in vitro and in silico approaches are developed to completely replace animal testing.
Air pollution leads to inhalation of several pulmonary stimulants that includes particulate matter (PM), and gaseous substances contributing significantly to the development of chronic lung diseases. However, the pathophysiological mechanism of air pollutant mediated pulmonary toxicity remains unclear. This is primarily due to the lack of efficient test systems, mimicing human inhalation exposure scenarios to air pollutants. The majority of the pulmonary in vitro-studies have been conducted using cell lines in submerged cell culture conditions and thereby overlooking the pulmonary physiology. Moreover, submerged cell culture systems lack the possibility to measure effective dose measurements. Particle properties, such as size, surface charge, solubility, transformation or agglomeration state and chemical properties are altered in solution and are dependent on the composition of cell culture medium. Physiologically relevant in vivo-like in vitro models cultured at air-liquid interface (ALI) is therefore becoming a realistic and efficient tool for lung toxicity testing and cell-cell interaction studies following exposure to aerosolized or gaseous form of air pollutants . Primary bronchial epithelial cells cultured at ALI leads to differentiate into respiratory epithelium consisting of ciliated cells, goblet cells, club cells and basal cells. ALI system is also considered as a feasible approach to implement the "3R principle"- replacement, reduction and refinement of animal usage in lung toxicity studies. This review discusses the current understanding of relevance, benefits and limitations of the ALI models in comparison to the existing in vitro and in vivo exposure system for testing air pollutants mediated pulmonary toxicity.
Organs-on-a-chip (OOCs) are miniature tissues and organs grown in vitro that enable modeling of human physiology and disease. The technology has emerged from converging advances in tissue engineering, semiconductor fabrication, and human cell sourcing. Encompassing innovations in human stem cell technology, OOCs offer a promising approach to emulate human patho/physiology in vitro, and address limitations of current cell and animal models. Here, we review the design considerations for single and multi-organ OOCs, discuss remaining challenges, and highlight the potential impact of OOCs as a fast-track opportunity for tissue engineering to advance drug development and precision medicine.
This paper presents a new type of cell culture membranes engineered from native extracellular matrix (ECM) materials that are thin, semipermeable, optically transparent, and amenable to integration into microfluidic cell culture devices.
This chapter reviews technical issues related to the toxicological testing of inhaled materials. The purpose of inhalation toxicology testing is to conduct studies in animals that will aid the assessment of the toxic potential of chemicals inhaled by humans. These chemicals may be related to environmental, occupational, and/or therapeutic concerns. Inhalation toxicity testing shares similar guiding principles with general toxicity testing. However, there are some unique issues, particularly with respect to delivery methods and dose of inhaled products, that need special attention when considering inhalation toxicology studies.
A new prototype air-liquid interface (ALI) exposure system, a flatbed aerosol exposure chamber termed NAVETTA, was developed to investigate deposition of engineered nanoparticles (NPs) on cultured human lung A549 cells directly from the gas phase. This device mimics human lung cell exposure to NPs due to a low horizontal gas flow combined with cells exposed at the ALI. Electrostatic field assistance is applied to improve NP deposition efficiency. As proof-of-principle, cell viability and immune responses after short-term exposure to nano-copper oxide (CuO)-aerosol were determined. We found that, due to the laminar aerosol flow and a specific orientation of inverted transwells, much higher deposition rates were obtained compared to the normal ALI setup. Cellular responses were monitored with post-exposure incubation in submerged conditions, revealing CuO dissolution in a concentration-dependent manner. Cytotoxicity was the result of ionic and non-ionic Cu fractions. Using the optimized inverted ALI/post-incubation procedure, pro-inflammatory immune responses, in terms of interleukin (IL)-8 promoter and nuclear factor kappa B (NFB) activity, were observed within short time, i.e. 1 hour exposure to ALI-deposited CuO-NPs and 2.5 hours post-incubation. NAVETTA is a novel option for mimicking human lung cell exposure to NPs, complementing existing ALI systems.
Smoking represents a major risk factor for chronic obstructive pulmonary disease (COPD), but it is difficult to characterize smoke-induced injury responses under physiological breathing conditions in humans due to patient-to-patient variability. Here, we show that a small airway-on-a-chip device lined by living human bronchiolar epithelium from normal or COPD patients can be connected to an instrument that “breathes” whole cigarette smoke in and out of the chips to study smoke-induced pathophysiology in vitro. This technology enables true matched comparisons of biological responses by culturing cells from the same individual with or without smoke exposure. These studies led to identification of ciliary micropathologies, COPD-specific molecular signatures, and epithelial responses to smoke generated by electronic cigarettes. The smoking airway-on-a-chip represents a tool to study normal and disease-specific responses of the human lung to inhaled smoke across molecular, cellular and tissue-level responses in an organ-relevant context.