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“Lung-on-a-chip” (LoC) is a microfluidic device, imitating the gas-fluid interface of the pulmonary alveole in the human lung and intended for pathophysiological, pharmacological and molecular-biological studies of the air-blood barrier in vitro. The LoC device itself contains a system of fluid and gas microchannels, separated with a semipermeable elastic membrane, containing a polymer base and the alveolar cell elements. Depending on the type of LoC (single-, double- and three-channel), the membrane may contain only alveolocytes or alveolocytes combined with other cells — endotheliocytes, fibroblasts, alveolar macrophages or tumor cells. Some LoC models also include proteinic or hydrogel stroma, imitating the pulmonary interstitium. The first double-channel LoC variant, in which one side of the membrane contained an alveolocytic monolayer and the other side — a monolayer of endotheliocytes, was developed in 2010 by a group of scientists from the Harvard University for maximally precise in vitro reproduction of the micro-environment and biomechanics operations of the alveoli. Modern LoC modifications include the same elements and differ only by the construction of the microfluidic system, by the biomaterial of semipermeable membrane, by the composition of cellular and stromal elements and by specific tasks to be solved. Besides the LoC imitating the hematoalveolar barrier, there are modifications for studying the specific pathophysiological processes, for the screening of medicinal products, for modeling specific diseases, for example, lung cancer, chronic obstructive pulmonary disease or asthma. In the present review, we have analyzed the existing types of LoC, the biomaterials used, the methods of detecting molecular processes within the microfluidic devices and the main directions of research to be conducted using the “lung-on-a-chip”.

Idiopathic pulmonary fibrosis (IPF) is a lethal disorder characterized by relentless progression of lung fibrosis that causes respiratory failure and early death. Currently, no curative treatments are available, and existing therapies include a limited selection of antifibrotic agents that only slow disease progression. The development of novel therapeutics has been hindered by a limited understanding of the disease's etiology and pathogenesis. A significant challenge in developing new treatments and understanding IPF is the lack of in vitro models that accurately replicate crucial microenvironments. In response, three-dimensional (3D) in vitro models have emerged as powerful tools for replicating organ-level microenvironments seen in vivo. This review summarizes the state of the art in advanced 3D lung models that mimic many physiological and pathological processes observed in IPF. We begin with a brief overview of conventional models, such as 2D cell cultures and animal models, and then explore more advanced 3D models, focusing on lung-on-a-chip systems. We discuss the current challenges and future research opportunities in this field, aiming to advance the understanding of the disease and the development of novel devices to assess the effectiveness of new IPF treatments. [ABSTRACT FROM AUTHOR]

Conventional dogma suggests that decompression sickness (DCS) is caused by nitrogen bubble nucleation in the blood vessels and/or tissues; however, the abundance of bubbles does not correlate with DCS severity. Since immune cells respond to chemical and environmental cues, we hypothesized that the elevated partial pressures of dissolved gases drive aberrant immune cell phenotypes in the alveolar vasculature. To test this hypothesis, we measured immune responses within human lung‐on‐a‐chip devices established with primary alveolar cells and microvascular cells. Devices were pressurized to 1.0 or 3.5 atm and surrounded by normal alveolar air or oxygen‐reduced air. Phenotyping of neutrophils, monocytes, and dendritic cells as well as multiplexed ELISA revealed that immune responses occur within 1 h and that normal alveolar air (i.e., hyperbaric oxygen and nitrogen) confer greater immune activation. This work strongly suggests innate immune cell reactions initiated at elevated partial pressures contribute to the etiology of DCS. [ABSTRACT FROM AUTHOR]

The physiological function of lung is strongly correlated with its unique structural microenvironment and mechanical stimulation. Most existing lung-on-a-chips (LOCs) do not replicate the key physiological structure and stimulation of human lung, reducing their reliability in application. In this study, a scaffold structure of a styrene-butadiene-styrene (SBS) nanofiber and porous honeycomb polydime-thylsiloxane (PDMS) composite membrane was developed to construct an alveolar air-blood barrier that mimics the alveolar characteristics of flexibility, cross-scale structure, and triaxial mechanical stimulation. By combining micro-fluidic and electrospinning technology, a biomimetic LOC with dynamic triaxial cyclic strain was realized. The composite membrane had a Young's modulus of 0.54 ± 0.05 MPa and was capable of 8–12% strain at 1 kPa air pressure. We monocultured and co-cultured human non-small cell lung cancer cells stably expressing red fluorescent protein (A549-RFP) with human umbilical vein endothelial cell stably expressing green fluorescent protein (HUVECs-GFP) within the chip. A multi-layered structure of epithelial cell layer-basal layer-endothelial cell layer, similar to the air-blood barrier in vivo, was constructed. The LOC was proved to be an initial foundation for creating in vitro alveolar physiological models, and could be a potential platform for application in physiology, pathology, toxicology, drug screening, and customized medicine. [ABSTRACT FROM AUTHOR]

Lung cancer is a malignant tumour with the highest incidence and mortality worldwide. Clinically effective therapy strategies are underutilized owing to the lack of efficient models for evaluating drug response. One of the main reasons for failure of anticancer drug therapy is development of drug resistance. Anticancer drugs face severe challenges such as poor biodistribution, restricted solubility, inadequate absorption, and drug accumulation. In recent years, “organ-on-a-chip” platforms, which can directly regulate the microenvironment of biomechanics, biochemistry and pathophysiology, have been developed rapidly and have shown great potential in clinical drug research. Lung-on-a-chip (LOC) is a new 3D model of bionic lungs with physiological functions created by micromachining technology on microfluidic chips. This approach may be able to partially replace animal and 2D cell culture models. To overcome drug resistance, LOC realizes personalized prediction of drug response by simulating the lung-related microenvironment in vitro, significantly enhancing therapeutic effectiveness, bioavailability, and pharmacokinetics while minimizing side effects. In this review, we present an overview of recent advances in the preparation of LOC and contrast it with earlier in vitro models. Finally, we describe recent advances in LOC. The combination of this technology with nanomedicine will provide an accurate and reliable treatment for preclinical evaluation.

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host–pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air–liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae , and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models. [ABSTRACT FROM AUTHOR]

Human organs‐on‐chips (organ chips) are small microfluidic devices that allow human cells to perform complex organ‐level functions in vitro by recreating multi‐cellular and multi‐tissue structures and applying in vivo‐like biomechanical cues. Human Organ Chips are being used for drug discovery and toxicology testing as an alternative to animal models which are ethically challenging and often do not predict clinical efficacy or toxicity. In this mini‐review, we summarize our presentation that reviewed the state of the art relating to these microfluidic culture devices designed to mimic specific human organ structures and functions, and the application of Organ Chips to regenerative pharmacology. [ABSTRACT FROM AUTHOR]

Lung cancer has become the primary cause of cancer-related deaths because of its high recurrence rate, ability to metastasise easily, and propensity to develop drug resistance. The wide-ranging heterogeneity of lung cancer subtypes increases the complexity of developing effective therapeutic interventions. Therefore, personalised diagnostic and treatment strategies are required to guide clinical practice. The advent of innovative three-dimensional (3D) culture systems such as organoid and organ-on-a-chip models provides opportunities to address these challenges and revolutionise lung cancer research and drug evaluation. In this review, we introduce the advancements in lung-related 3D culture systems, with a particular focus on lung organoids and lung-on-a-chip, and their latest contributions to lung cancer research and drug evaluation. These developments include various aspects, from authentic simulations and mechanistic enquiries into lung cancer to assessing chemotherapeutic agents and targeted therapeutic interventions. The new 3D culture system can mimic the pathological and physiological microenvironment of the lung, enabling it to supplement or replace existing two-dimensional culture models and animal experimental models and realize the potential for personalised lung cancer treatment.

The global burden of respiratory diseases is enormous, with many millions of people suffering and dying prematurely every year. The global COVID‐19 pandemic witnessed recently, along with increased air pollution and wildfire events, increases the urgency of identifying the most effective therapeutic measures to combat these diseases even further. Despite increasing expenditure and extensive collaborative efforts to identify and develop the most effective and safe treatments, the failure rates of drugs evaluated in human clinical trials are high. To reverse these trends and minimize the cost of drug development, ineffective drug candidates must be eliminated as early as possible by employing new, efficient, and accurate preclinical screening approaches. Animal models have been the mainstay of pulmonary research as they recapitulate the complex physiological processes, Multiorgan interplay, disease phenotypes of disease, and the pharmacokinetic behavior of drugs. Recently, the use of advanced culture technologies such as organoids and lung‐on‐a‐chip models has gained increasing attention because of their potential to reproduce human diseased states and physiology, with clinically relevant responses to drugs and toxins. This review provides an overview of different animal models for studying respiratory diseases and evaluating drugs. We also highlight recent progress in cell culture technologies to advance integrated models and discuss current challenges and present future perspectives. [ABSTRACT FROM AUTHOR]

Lung‐on‐chips have showed great promise as a tool to recapitulate the respiratory system for investigation of lung diseases in the past decade. However, the commonly applied artificial elastic membrane (e.g., polydimethylsiloxane, PDMS) in the chip failed to mimic the alveolar basal membrane in the composition and mechanical properties. Here we replaced the PDMS film by a thin, biocompatible, soft, and stretchable membrane based on F127‐DA hydrogel that well approached to the composition and stiffness of extracellular matrix in human alveoli for construction of lung‐on‐a‐chip. This chip well reconstructed the mechanical microenvironments in alveoli so that the epithelial/endothelial functions were highly expressed with a well established alveolar‐capillary barrier. In opposite to the unexpectedly accelerated fibrotic process on the PDMS‐based lung‐on‐a‐chip, HPAEpiCs on hydrogel‐based chip only presented fibrosis under nonphysiologically high strain, well reflecting the features of pulmonary fibrosis in vivo. This physiologically relevant lung‐on‐a‐chip would be an ideal model in investigation of lung diseases and for development of antifibrosis drugs. [ABSTRACT FROM AUTHOR]

Lung diseases have become a significant challenge to public healthcare worldwide, which stresses the necessity of developing effective biological models for pathophysiological and pharmacological studies of the human respiratory system. In recent years, lung-on-a-chip has been extensively developed as a potentially revolutionary respiratory model paradigm with high efficiency and improved accuracy, bridging the gap between cell culture and preclinical trials. The advantages of lung-on-a-chip technology derive from its capabilities in establishing 3D multicellular architectures and dynamic microphysiological environments. A critical issue in its development is utilizing such capabilities to recapitulate the essential components of the human respiratory system for effectively restoring physiological functions and illustrating disease progress. Here we present a review of lung-on-a-chip technology, highlighting various strategies for capturing lung physiological and pathological characteristics. The key pathophysiological characteristics of the lungs are examined, including the airways, alveoli, and alveolar septum. Accordingly, the strategies in lung-on-a-chip research to capture the essential components and functions of lungs are analyzed. Recent studies of pneumonia, lung cancer, asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis based on lung-on-a-chip are surveyed. Finally, cross-disciplinary approaches are proposed to foster the future development of lung-on-a chip technology. [ABSTRACT FROM AUTHOR]

The incidence of respiratory diseases such as chronic obstructive pulmonary disease and pulmonary cancer is growing significantly around the world, making pulmonary disease one of the leading causes of mortality. However, the development of effective therapeutics for pulmonary diseases has been hindered by the lack of human‐mimetic physiological models that reliably emulate patient responses. Recent advances in technology and cell culture have led to the development of organoids and organ‐on‐a‐chip models that allow us to recapitulate the structure, cellular organization, and organ‐level responses of the target tissue in vitro. Here, we review the advances and milestones of lung organoid and lung‐on‐a‐chip models in the past decade and highlight their applications in mimicking pulmonary system development, physiology, disease, and regeneration. In addition, we discuss the ongoing challenges and the future prospects of integrating lung organoids and lung‐on‐a‐chip models to overcome current limitations and to enhance their physiological relevance. These human‐centric models are likely to provide important insights into pulmonary physiology and pathophysiology for drug discovery that complement and potentially replace traditional animal models. [ABSTRACT FROM AUTHOR]

Intricate crosstalk among various lung cell types is crucial for orchestrating diverse physiological processes. Traditional two-dimensional and recent three-dimensional (3D) assay platforms fail to precisely replicate these complex communications. Many in vitro lung models do not effectively reflect the multicellular complexity of lung tissue. Here, we fabricated an advanced multicellular 3D lung-on-a-chip system that properly replicates the dynamic pulmonary microenvironment and its intricate microarchitecture. Diverse lung cells were incorporated into a microstructure formed from a mixture of natural polymers, including collagen and hyaluronic acid, and blood coagulation factors acting as natural crosslinking agents. The system accurately reflects the complex 3D architecture of the lung. Biomarkers demonstrate more rapid and sensitive responses to toxic substances than functional indicators, such as cell proliferation and apoptosis. SERPINB2 was identified as a biomarker of lung toxicity; it was activated in small airway epithelial cells exposed to various toxic substances. We then developed a fluorescence-linked toxicity biomarker screening platform that enables both intuitive and quantitative evaluation of lung toxicity by measuring the converted fluorescent signal strength. This fluorescent tagging system was incorporated into small airway epithelial cells within a fabricated chip platform; enabling lung-on-a-chip enabled evaluation of the lung toxicity of prospective drug candidates., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Abstract The lung is the respiratory organ of the human body, and the alveoli are the most basic functional units of the lung. Herein, a photo‐responsive stretchable Janus membrane was proposed for the reconstruction of the alveolar–capillary barrier in vitro. This Janus membrane was fabricated by photocrosslinking methylacrylamide gelatin (Gelma) hydrogel and N‐isoacrylamide (NIPAM) hydrogel mixed with graphene oxide (GO). The Gelma hydrogel containing large amounts of collagen provides a natural extracellular matrix environment for cell growth, while the temperature‐sensitive NIPAM hydrogel combined with GO gives the membrane a light‐controlled stretching property. Based on this Janus membrane, an open polydimethylsiloxane chip was established to coculture alveolar epithelial cells and vascular endothelial cells at the air–liquid interface. It was demonstrated that the alveolar epithelial cells cultured on the upper side of the Janus membrane could express epithelial cell marker protein E‐cadherin and secrete alveolar surfactant. In addition, VE‐cadherin, an endothelium‐specific protein located at the junction between endothelial cells, was also detected in vascular endothelial cells cultured on the underside of Janus membrane. The constructed lung tissue model with the dynamically stretchable Janus membrane is well‐suited for COVID‐19 infection studies and drug testing.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a never before seen challenge to human health and the world economy. However, it is difficult to widely use conventional animal and cell culture models in understanding the underlying pathological mechanisms of COVID-19, which in turn hinders the development of relevant therapeutic treatments, including drugs. To overcome this challenge, various three-dimensional (3D) pulmonary cell culture models such as organoids are emerging as an innovative toolset for simulating the pathophysiology occurring in the respiratory system, including bronchial airways, alveoli, capillary network, and pulmonary interstitium, which provide a robust and powerful platform for studying the process and underlying mechanisms of SARS-CoV-2 infection among the potential primary targets in the lung. This review introduces the key features of some of these recently developed tools, including organoid, lung-on-a-chip, and 3D bioprinting, which can recapitulate different structural compartments of the lung and lung function, in particular, accurately resembling the human-relevant pathophysiology of SARS-CoV-2 infection in vivo. In addition, the recent progress in developing organoids for alveolar and airway disease modeling and their applications for discovering drugs against SARS-CoV-2 infection are highlighted. These innovative 3D cell culture models together may hold the promise to fully understand the pathogenesis and eventually eradicate the pandemic of COVID-19. [ABSTRACT FROM AUTHOR]

Recently, there has been increasing attention toward inhaled nanoparticles (NPs) to develop inhalation therapies for diseases associated with the pulmonary system and investigate the toxic effects of hazardous environmental particles on human lung health. Taking advantage of microfluidic technology for cell culture applications, lung-on-a-chip devices with great potential in replicating the lung air–blood barrier (ABB) have opened new research insights in preclinical pathology and therapeutic studies associated with aerosol NPs. However, the air interface in such devices has been largely disregarded, leaving a gap in understanding the NPs' dynamics in lung-on-a-chip devices. Here, we develop a numerical parametric study to provide insights into the dynamic behavior of the airborne NPs in a gas–liquid dual-channel lung-on-a-chip device with a porous membrane separating the channels. We develop a finite element multi-physics model to investigate particle tracing in both air and medium phases to replicate the in vivo conditions. Our model considers the impact of fluid flow and geometrical properties on the distribution, deposition, and translocation of NPs with diameters ranging from 10 nm to 900 nm. Our findings suggest that, compared to the aqueous solution of NPs, the aerosol injection of NPs offers more efficient deposition on the substrate of the air channel and higher translocation to the media channel. Comparative studies against accessible data, as well as an experimental study, verify the accuracy of the present numerical analysis. We propose a strategy to optimize the affecting parameters to control the injection and delivery of aerosol particles into the lung-on-chip device depending on the objectives of biomedical investigations and provide optimized values for some specific cases. Therefore, our study can assist scientists and researchers in complementing their experimental investigation in future preclinical studies on pulmonary pathology associated with inhaled hazardous and toxic environmental particles, as well as therapeutic studies for developing inhalation drug delivery. [ABSTRACT FROM AUTHOR]

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and antiinflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrixderived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a betteradjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy. [ABSTRACT FROM AUTHOR]

With the global burden of respiratory diseases, rapid identification of the best therapeutic measures to combat these diseases is essential. Animal models and 2D cell culture models do not replicate the findings observed in vivo. To gain deeper insight into lung pathology and physiology, 3D and advanced lung-on-a-chip models have been developed recently. Lung-on-a-chip models more accurately simulate the lung's microenvironment and functions in vivo , resulting in more-accurate assessments of drug safety and effectiveness. This review discusses the transition from 2D to 3D models and the recent advances in lung-on-a-chip platforms, their implementation and the numerous challenges faced. Finally, a general overview of this platform and its potential applications in respiratory disease research and drug discovery is highlighted. • Transition from conventional 2D models to 3D and advanced lung-on-a-chip models. • Recent advances in lung-on-a-chip platforms and their implementation. • Numerous challenges faced in lung-on-a-chip devices. • Potential applications in respiratory disease research and drug discovery. [ABSTRACT FROM AUTHOR]

In this study, we used three-dimensional (3D) printing to prepare a template of a microfluidic chip from which a polydimethylsiloxane (PDMS)lung chip was successfully constructed. The upper and lower channels of the chip are separated by a microporous membrane. The upper channel is seeded with lung cancer cells, and the lower channel is seeded with vascular endothelial cells and continuously perfused with cell culture medium. This lung chip can simulate the microenvironment of lung tissue and realize the coculture of two kinds of cells at different levels. We used a two-dimensional (2D) well plate and a 3D lung chip to evaluate the effects of different EGFR-targeting drugs (gefitinib, afatinib, and osimertinib) on tumor cells. The 3D lung chip was superior to the 2D well plate at evaluating the effect of drugs on the NCI-H650, and the results were more consistent with existing clinical data. For primary tumor cells, 3D lung chips have more advantages because they simulate conditions that are more similar to the physiological cell microenvironment. The evaluation of EGFR-targeted drugs on lung chips is of great significance for personalized diagnosis and treatment and pharmacodynamic evaluation. [ABSTRACT FROM AUTHOR]

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy.

In this work, we present a programmable dynamic cell culture stimulation platform designed to work in a CO2 incubator, compatible with a standard multi-well plate. An epithelial lung cell culture can be mechanically stimulated with a cyclic increase of the hydrostatic pressure inside the culture chamber in the range 1–12 kPa, trying to mimic the human respiration rate. An in situ live pH-monitoring system based on PEDOT:PSS Organic ElectroChemical Transistors was also designed, fabricated and characterized, in order to merge both the stimulated cell culture and the pH sensing in a compact platform.

In end-stage lung diseases, the shortage of donor lungs for transplantation and long waiting lists are the main culprits in the significantly increasing number of patient deaths. New strategies to curb this issue are being developed with the help of recent advancements in bioengineering technology, with the generation of lung scaffolds as a steppingstone. There are various types of lung scaffolds, namely, acellular scaffolds that are developed via decellularization and recellularization techniques, artificial scaffolds that are synthesized using synthetic, biodegradable, and low immunogenic materials, and hybrid scaffolds which combine the advantageous properties of materials in the development of a desirable lung scaffold. There have also been advances in the design of bioreactors in terms of providing an optimal regenerative environment for the maturation of functional lung tissue over time. In this review, the emerging paradigms in the field of lung tissue bioengineering will be discussed. [ABSTRACT FROM AUTHOR]

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 evaluation of inhalation toxicity, drug safety and efficacy assessment, as well as the investigation of complex disease pathomechanisms, are increasingly relying on in vitro lung models. This is due to the progressive shift towards human-based systems for more predictive and translational research. While several cellular models are currently available for the upper airways, modelling the distal alveolar region poses several constraints that make the standardization of reliable alveolar in vitro models relatively difficult. In this work, we present a new and reproducible alveolar in vitro model, that combines a human derived immortalized alveolar epithelial cell line (AXiAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment (AXlung-on-chip). The latter mimics key features of the in vivo alveolar milieu: breathing-like 3D cyclic stretch (10% linear strain, 0.2 Hz frequency) and an ultrathin, porous and elastic membrane. AXiAECs cultured on-chip were characterized for their alveolar epithelial cell markers by gene and protein expression. Cell barrier properties were examined by TER (Transbarrier Electrical Resistance) measurement and tight junction formation. To establish a physiological model for the distal lung, AXiAECs were cultured for long-term at air-liquid interface (ALI) on-chip. To this end, different stages of alveolar damage including inflammation (via exposure to bacterial lipopolysaccharide) and the response to a profibrotic mediator (via exposure to Transforming growth factor β1) were analyzed. In addition, the expression of relevant host cell factors involved in SARS-CoV-2 infection was investigated to evaluate its potential application for COVID-19 studies. This study shows that AXiAECs cultured on the AXlung-on-chip exhibit an enhanced in vivo-like alveolar character which is reflected into: 1) Alveolar type 1 (AT1) and 2 (AT2) cell specific phenotypes, 2) tight barrier formation (with TER above 1,000 Ω cm2) and 3) reproducible long-term preservation of alveolar characteristics in nearly physiological conditions (co-culture, breathing, ALI). To the best of our knowledge, this is the first time that a primary derived alveolar epithelial cell line on-chip representing both AT1 and AT2 characteristics is reported. This distal lung model thereby represents a valuable in vitro tool to study inhalation toxicity, test safety and efficacy of drug compounds and characterization of xenobiotics.

Here, we present a bioengineering approach to emulate the human bone marrow in vitro. Our developmentally inspired method uses self-organization of human hematopoietic stem and progenitor cells and vascular endothelial cells cultured in a three-dimensional microphysiological system to create vascularized, perfusable tissue constructs that resemble the hematopoietic vascular niche of the human marrow. The microengineered niche is capable of multilineage hematopoiesis and can generate functionally mature human myeloid cells that can intravasate into perfused blood vessels, providing a means to model the mobilization of innate immune cells from the marrow. We demonstrate the application of this system by presenting a specialized model of ionizing radiation-induced bone marrow injury and a multiorgan model of acute innate immune responses to bacterial lung infection. Furthermore, we introduce an advanced platform that enables large-scale integration and automated experimentation of the engineered hematopoietic tissues for preclinical screening of myelotoxicity due to anti-cancer drugs., Competing Interests: Declaration of interests A.G., J.H.G., T.C., and D.D.H. hold equity in Vivodyne Inc. A.G. and D.D.H. are co-founders, employees, and board directors of Vivodyne Inc. J.H.G., T.C., and P.L.C. are employees of Vivodyne Inc. A.L. is an employee of GSK and holds equity in the company. A patent application describing a technology related to this work is currently under review that includes A.G., G.S.W., and D.D.H. as co-inventors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human organ-on-a-chip models are powerful tools for preclinical research that can be used to study the mechanisms of disease and evaluate new targets for therapeutic intervention. Lung-on-a-chip models have been one of the most well-characterized designs in this field and can be altered to evaluate various types of respiratory disease and to assess treatment candidates prior to clinical testing. These systems are capable of overcoming the flaws of conventional two-dimensional (2-D) cell culture and in vivo animal testing due to their ability to accurately recapitulate the in vivo microenvironment of human tissue with tunable material properties, microfluidic integration, delivery of precise mechanical and biochemical cues, and designs with organ-specific architecture. In this review, we first describe an overview of currently available lung-on-a-chip designs. We then present how recent innovations in human stem cell biology, tissue engineering, and microfabrication can be used to create more predictive human lung-on-a-chip models for studying respiratory disease. Finally, we discuss the current challenges and future directions of lung-on-a-chip designs for in vitro disease modeling with a particular focus on immune and multiorgan interactions. [ABSTRACT FROM AUTHOR]

Microfluidic lung‐on‐a‐chip systems are increasingly attractive tools for studying lung physiology and function because of their ability to accurately recapitulate spatiotemporal features of the airway tissue microenvironment including cellular organization, tissue architecture, and mechanical cues such as cyclic stretching and airflow. However, most lung‐on‐a‐chip devices to date rely on integrated design elements like membranes for airway cell culture, and focus mainly on enabling on‐chip monitoring and analysis while neglecting the need for off‐chip analysis. Here, an extractable floating liquid‐gel‐based organ‐on‐a‐chip for airway tissue modeling referred to as "E‐FLOAT" is described that is arrayable, scalable, and uniquely amenable to withstand physiologic airflow by microanchors. It is shown that E‐FLOAT can be combined with a custom airflow system that permits controlled injection of particulate matter for air pollution studies. Results show that airflow is critical to efficiently achieving physiologic mimicry of airway epithelium composition, tight junction expression, mucus production, and cilia formation on epithelial cells. It is also shown that E‐FLOAT allows standard on‐chip analysis while permitting complete sample extraction and off‐chip analysis via immunocytochemistry, microscopy, and histological sectioning and staining, thereby expanding the number and types of biological assays that can be used and questions that can be tackled. [ABSTRACT FROM AUTHOR]

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research. [Display omitted] [ABSTRACT FROM AUTHOR]

Recently, there has been increasing attention toward inhaled nanoparticles (NPs) to develop inhalation therapies for diseases associated with the pulmonary system and investigate the toxic effects of hazardous environmental particles on human lung health. Taking advantage of microfluidic technology for cell culture applications, lung-on-a-chip devices with great potential in replicating the lung air–blood barrier (ABB) have opened new research insights in preclinical pathology and therapeutic studies associated with aerosol NPs. However, the air interface in such devices has been largely disregarded, leaving a gap in understanding the NPs’ dynamics in lung-on-a-chip devices. Here, we develop a numerical parametric study to provide insights into the dynamic behavior of the airborne NPs in a gas–liquid dual-channel lung-on-a-chip device with a porous membrane separating the channels. We develop a finite element multi-physics model to investigate particle tracing in both air and medium phases to replicate the in vivo conditions. Our model considers the impact of fluid flow and geometrical properties on the distribution, deposition, and translocation of NPs with diameters ranging from 10 nm to 900 nm. Our findings suggest that, compared to the aqueous solution of NPs, the aerosol injection of NPs offers more efficient deposition on the substrate of the air channel and higher translocation to the media channel. Comparative studies against accessible data, as well as an experimental study, verify the accuracy of the present numerical analysis. We propose a strategy to optimize the affecting parameters to control the injection and delivery of aerosol particles into the lung-on-chip device depending on the objectives of biomedical investigations and provide optimized values for some specific cases. Therefore, our study can assist scientists and researchers in complementing their experimental investigation in future preclinical studies on pulmonary pathology associated with inhaled hazardous and toxic environmental particles, as well as therapeutic studies for developing inhalation drug delivery.

In this study, we used three-dimensional (3D) printing to prepare a template of a microfluidic chip from which a polydimethylsiloxane (PDMS)lung chip was successfully constructed. The upper and lower channels of the chip are separated by a microporous membrane. The upper channel is seeded with lung cancer cells, and the lower channel is seeded with vascular endothelial cells and continuously perfused with cell culture medium. This lung chip can simulate the microenvironment of lung tissue and realize the coculture of two kinds of cells at different levels. We used a two-dimensional (2D) well plate and a 3D lung chip to evaluate the effects of different EGFR-targeting drugs (gefitinib, afatinib, and osimertinib) on tumor cells. The 3D lung chip was superior to the 2D well plate at evaluating the effect of drugs on the NCI-H650, and the results were more consistent with existing clinical data. For primary tumor cells, 3D lung chips have more advantages because they simulate conditions that are more similar to the physiological cell microenvironment. The evaluation of EGFR-targeted drugs on lung chips is of great significance for personalized diagnosis and treatment and pharmacodynamic evaluation.

Lung bioengineering has emerged to resolve the current lung transplantation limitations and risks, including the shortage of donor organs and the high rejection rate of transplanted lungs. One of the most critical elements of lung bioengineering is bioreactors. Bioreactors with different applications have been developed in the last decade for lung bioengineering approaches, aiming to produce functional reproducible tissue constructs. Here, the current status and advances made in the development and application of bioreactors for bioengineering lungs are comprehensively reviewed. First, bioreactor design criteria are explained, followed by a discussion on using bioreactors as a culture system for scalable expansion and proliferation of lung cells, such as producing epithelial cells from induced pluripotent stem cells (iPSCs). Next, bioreactor systems facilitating and improving decellularization and recellularization of lung tissues are discussed, highlighting the studies that developed bioreactors for producing engineered human‐sized lungs. Then, monitoring bioreactors are reviewed, showing their ability to evaluate and optimize the culture conditions for maturing engineered lung tissues, followed by an explanation on the ability of ex vivo lung perfusion systems for reconditioning the lungs before transplantation. After that, lung cancer studies simplified by bioreactors are discussed, showing the potentials of bioreactors in lung disease modeling. Finally, other platforms with the potential of facilitating lung bioengineering are described, including the in vivo bioreactors and lung‐on‐a‐chip models. In the end, concluding remarks and future directions are put forward to accelerate lung bioengineering using bioreactors. [ABSTRACT FROM AUTHOR]

Here, we present a physiologically relevant model of the human pulmonary alveoli. This alveolar lung-on-a-chip platform is composed of a three-dimensional porous hydrogel made of gelatin methacryloyl with an inverse opal structure, bonded to a compartmentalized polydimethylsiloxane chip. The inverse opal hydrogel structure features well-defined, interconnected pores with high similarity to human alveolar sacs. By populating the sacs with primary human alveolar epithelial cells, functional epithelial monolayers are readily formed. Cyclic strain is integrated into the device to allow biomimetic breathing events of the alveolar lung, which, in addition, makes it possible to investigate pathological effects such as those incurred by cigarette smoking and severe acute respiratory syndrome coronavirus 2 pseudoviral infection. Our study demonstrates a unique method for reconstitution of the functional human pulmonary alveoli in vitro, which is anticipated to pave the way for investigating relevant physiological and pathological events in the human distal lung. [ABSTRACT FROM AUTHOR]

We report on the development of a new model of alveolar air–tissue interface on a chip. The model consists of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40–80 µm at the air–liquid interface by application of air pressure in the range of 200–1,000 Pa. With respect to the diameter of the monolayers, that is, 500 µm, this displacement corresponds to a linear strain of 2–10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time of 1–3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz for 1 hr to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy. [ABSTRACT FROM AUTHOR]

One of the key problems that have hindered the development and approval of anticancer nanoparticle drug delivery systems is the limited predictability of 2D cell culture and animal models. Here, we describe a biomimetic alveolus-epithelium-on-a-chip (AEOC) model with in-built sensors for monitoring and evaluating pH-responsive zinc oxide quantum dots (QDs)-loaded human serum albumin nanoparticles. This AEOC model closely represents the cancerous alveolus epithelium, which comprises lung cancer cells, as well as stromal cells, such as fibroblasts along with extracellular matrix (ECM) in the form of collagen. ZnO QDs were encapsulated in the HSA nanoparticles with a diameter of 60 nm. The physicochemical properties, quantum dots release, in vitro cytotoxicity, and cellular uptake of HSA-ZnO were evaluated. HSA-ZnO showed higher ZnO loading and encapsulation efficacy. TEER and pH sensors were used to monitor the cells over three days, and real-time data with and without nanoparticle treatment were obtained. Cell viability after treatment with 10 and 50 µg/mL of HSA-ZnO nanoparticles and confocal imaging data confirmed the significant internalization of the nanoparticles under co-culture cellular conditions in the AEOC model. Our designed organ-on-a-chip model has potentially expanded the capabilities of cell culture in biomimetic conditions, and therefore, can provide a low-cost alternative to expensive and tedious animal models for the evaluation of nanomedicines. [ABSTRACT FROM AUTHOR]

Lung diseases (e.g., infection, asthma, cancer, and pulmonary fibrosis) represent serious threats to human health all over the world. Conventional two-dimensional (2D) cell models and animal models cannot mimic the human-specific properties of the lungs. In the past decade, human organ-on-a-chip (OOC) platforms—including lung-on-a-chip (LOC)—have emerged rapidly, with the ability to reproduce the in vivo features of organs or tissues based on their three-dimensional (3D) structures. Furthermore, the integration of biosensors in the chip allows researchers to monitor various parameters related to disease development and drug efficacy. In this review, we illustrate the biosensor-based LOC modeling, further discussing the future challenges as well as perspectives in integrating biosensors in OOC platforms.

Organ-on-a-chip technology has the potential to accelerate pharmaceutical drug development, improve the clinical translation of basic research, and provide personalized intervention strategies. In the last decade, big pharma has engaged in many academic research cooperations to develop organ-on-a-chip systems for future drug discoveries. Although most organ-on-a-chip systems present proof-of-concept studies, miniaturized organ systems still need to demonstrate translational relevance and predictive power in clinical and pharmaceutical settings. This review explores whether microfluidic technology succeeded in paving the way for developing physiologically relevant human in vitro models for pharmacology and toxicology in biomedical research within the last decade. Individual organ-on-a-chip systems are discussed, focusing on relevant applications and highlighting their ability to tackle current challenges in pharmacological research.

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic.