Reversed-engineered human alveolar lung-on-a-chip model.

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.

Advanced models for respiratory disease and drug studies.

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.

A microfluidic lung-on-a-chip based on biomimetic hydrogel membrane.

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.

Engineering Dynamic 3D Models of Lung.

The lung parenchyma-consisting of gas-filled alveoli, vasculature, and connective tissue-is the site for gas exchange in the lung and plays a critical role in a number of chronic lung diseases. In vitro models of lung parenchyma can, therefore, provide valuable platforms for the study of lung biology in health and disease. Yet modeling such a complex tissue requires integrating multiple components, including biochemical cues from the extracellular environment, geometrically defined multicellular interactions, and dynamic mechanical inputs such as the cyclic stretch of breathing. In this chapter, we provide an overview of the broad spectrum of model systems that have been developed to recapitulate one or more features of lung parenchyma, and some of the scientific advances generated by those models. We discuss the use of both synthetic and naturally derived hydrogel materials, precision-cut lung slices, organoids, and lung-on-a-chip devices, with perspectives on the strengths, weaknesses, and potential future directions of these engineered systems.

Understanding and Engineering the Pulmonary Vasculature.

Blood vessels play essential roles in regulating embryonic organogenesis and adult tissue homeostasis. The inner lining of blood vessels is covered by vascular endothelial cells, which exhibit tissue-specific phenotypes in term of their molecular signature, morphology, and function. The pulmonary microvascular endothelium is continuous and non-fenestrae to ensure stringent barrier function while allowing efficient gas exchange across the alveoli-capillary interface. During respiratory injury repair, pulmonary microvascular endothelial cells secrete unique angiocrine factors and actively participate in the molecular and cellular events mediating alveolar regeneration. Advances in stem cell and organoid engineering are offering new ways to produce vascularized lung tissue models to investigate vascular-parenchymal interactions during lung organogenesis and pathogenesis. Further, technology developments in 3D biomaterial fabrication are enabling construction of vascularized tissues and microdevices with organotypic features at high resolution to recapitulate the air-blood interface. In parallel, whole-lung decellularization produces biomaterial scaffolds with naturally occurring, acellular vascular bed with preserved tissue architecture and complexity. Emerging efforts in combining cells with synthetic or natural biomaterials open vast opportunities for engineering the organotypic pulmonary vasculature to address current limitations in regenerating and repairing damaged lungs and pave the way towards next-generation therapies for pulmonary vascular diseases.

Three-dimensional models of the lung: past, present and future: a mini review.

Respiratory diseases are a major reason for death in both men and women worldwide. The development of therapies for these diseases has been slow and the lack of relevant human models to understand lung biology inhibits therapeutic discovery. The lungs are structurally and functionally complex with many different cell types which makes designing relevant lung models particularly challenging. The traditional two-dimensional (2D) cell line cultures are, therefore, not a very accurate representation of the in vivo lung tissue. The recent development of three-dimensional (3D) co-culture systems, popularly known as organoids/spheroids, aims to bridge the gap between 'in-dish' and 'in-tissue' cell behavior. These 3D cultures are modeling systems that are widely divergent in terms of culturing techniques (bottom-up/top-down) that can be developed from stem cells (adult/embryonic/pluripotent stem cells), primary cells or from two or more types of cells, to build a co-culture system. Lung 3D models have diverse applications including the understanding of lung development, lung regeneration, disease modeling, compound screening, and personalized medicine. In this review, we discuss the different techniques currently being used to generate 3D models and their associated cellular and biological materials. We further detail the potential applications of lung 3D cultures for disease modeling and advances in throughput for drug screening.

Human microphysiological models of airway and alveolar epithelia.

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.

Advances in bioreactors for lung bioengineering: From scalable cell culture to tissue growth monitoring.

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.

Lung-on-a-chip: the future of respiratory disease models and pharmacological studies.

Recently, organ-on-a-chip models, which are microfluidic devices that mimic the cellular architecture and physiological environment of an organ, have been developed and extensively investigated. The chips can be tailored to accommodate the disease conditions pertaining to many organs; and in the case of this review, the lung. Lung-on-a-chip models result in a more accurate reflection compared to conventional in vitro models. Pharmaceutical drug testing methods traditionally use animal models in order to evaluate pharmacological and toxicological responses to a new agent. However, these responses do not directly reflect human physiological responses. In this review, current and future applications of the lung-on-a-chip in the respiratory system will be discussed. Furthermore, the limitations of current conventional in vitro models used for respiratory disease modeling and drug development will be addressed. Highlights of additional translational aspects of the lung-on-a-chip will be discussed in order to demonstrate the importance of this subject for medical research.

Alveolar mimics with periodic strain and its effect on the cell layer formation.

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.

Progress and application of lung-on-a-chip for lung cancer.

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.

Evaluation of drug resistance for EGFR-TKIs in lung cancer via multicellular lung-on-a-chip.

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.

In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models.

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.

Human organ chips for regenerative pharmacology.

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.

Sentinel supervised lung-on-a-chip: A new environmental toxicology platform for nanoplastic-induced lung injury.

Nanoplastics are prevalent in the air and can be easily inhaled, posing a threat to respiratory health. However, there have been few studies investigating the impact of nanoplastics on lung injury, especially chronic obstructive pulmonary disease (COPD). Furthermore, cell and animal models cannot deeply understand the pollutant-induced COPD. Existing lung-on-a-chip models also lack interactions among immune cells, which are crucial in monitoring complex responses. In the study, we built the lung-on-a-chip to accurately recapitulate the structural features and key functions of the alveolar-blood barrier while integrating multiple immune cells. The stability and reliability of the lung-on-a-chip model were demonstrated by toxicological application of various environmental pollutants. We Further focused on exploring the association between COPD and polystyrene nanoplastics (PS-NPs). As a result, the cell viability significantly decreased as the concentration of PS-NPs increased, while TEER levels decreased and permeability increased. Additionally, PS-NPs could induce oxidative stress and inflammatory responses at the organ level, and crossed the alveolar-blood barrier to enter the bloodstream. The expression of α1-antitrypsin (AAT) was significantly reduced, which could be served as early COPD checkpoint on the lung-chips. Overall, the lung-on-a-chip provides a new platform for investigating the pulmonary toxicity of nanoplastics, demonstrating that PS-NPs can harm the alveolar-blood barrier, cause oxidative damage and inflammation, and increase the risk of COPD.

A storm in a teacup -- A biomimetic lung microphysiological system in conjunction with a deep-learning algorithm to monitor lung pathological and inflammatory reactions.

Creating a biomimetic in vitro lung model to recapitulate the infection and inflammatory reactions has been an important but challenging task for biomedical researchers. The 2D based cell culture models - culturing of lung epithelium - have long existed but lack multiple key physiological conditions, such as the involvement of different types of immune cells and the creation of connected lung models to study viral or bacterial infection between different individuals. Pioneers in organ-on-a-chip research have developed lung alveoli-on-a-chip and connected two lung chips with direct tubing and flow. Although this model provides a powerful tool for lung alveolar disease modeling, it still lacks interactions among immune cells, such as macrophages and monocytes, and the mimic of air flow and aerosol transmission between lung-chips is missing. Here, we report the development of an improved human lung physiological system (Lung-MPS) with both alveolar and pulmonary bronchial chambers that permits the integration of multiple immune cells into the system. We observed amplified inflammatory signals through the dynamic interactions among macrophages, epithelium, endothelium, and circulating monocytes. Furthermore, an integrated microdroplet/aerosol transmission system was fabricated and employed to study the propagation of pseudovirus particles containing microdroplets in integrated Lung-MPSs. Finally, a deep-learning algorithm was developed to characterize the activation of cells in this Lung-MPS. This Lung-MPS could provide an improved and more biomimetic sensory system for the study of COVID-19 and other high-risk infectious lung diseases.

Biomimetic lung-on-a-chip to model virus infection and drug evaluation.

Viral infectious diseases remain a global public health problem. The rapid and widespread spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV­2) has had a severe impact on the global economy and human activities, highlighting the vulnerability of humans to viral infectious diseases and the urgent need to develop new technologies and effective treatments. Organ-on-a-chip is an emerging technology for constructing the physiological and pathological microenvironment of human organs in vitro and has the advantages of portability, high throughput, low cost, and accurate simulation of the in vivo microenvironment. Indeed, organ-on-a-chip provides a low-cost alternative for investigating human organ physiology, organ diseases, toxicology, and drug efficacy. The lung is a main target organ of viral infection, and lung pathophysiology must be assessed after viral infection and treatment with antiviral drugs. This review introduces the construction of lung-on-a-chip and its related pathophysiological models, focusing on the in vitro simulation of viral infection and evaluation of antiviral drugs, providing a developmental direction for research and treatment of viral diseases.

Recapitulating essential pathophysiological characteristics in lung-on-a-chip for disease studies.

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.

Ferritinophagy Mediated by Oxidative Stress-Driven Mitochondrial Damage Is Involved in the Polystyrene Nanoparticles-Induced Ferroptosis of Lung Injury.

Nanoplastics are a common type of contaminant in the air. However, no investigations have focused on the toxic mechanism of lung injury induced by nanoplastic exposure. In the present study, polystyrene nanoplastics (PS-NPs) caused ferroptosis in lung epithelial cells, which could be alleviated by ferrostatin-1, deferoxamine, and N-acetylcysteine. Further investigation found that PS-NPs disturbed mitochondrial structure and function and triggered autophagy. Mechanistically, oxidative stress-derived mitochondrial damage contributed to ferroptosis, and autophagy-dependent ferritinophagy was a pivotal intermediate link, resulting in ferritin degradation and iron ion release. Furthermore, inhibition of ferroptosis using ferrostatin-1 alleviated pulmonary and systemic toxicity to reverse the mouse lung injury induced by PS-NPs inhalation. Most importantly, the lung-on-a-chip was further used to clarify the role of ferroptosis in the PS-NPs-induced lung injury by visualizing the ferroptosis, oxidative stress, and alveolar-capillary barrier dysfunction at the organ level. In summary, our study indicated that ferroptosis was an important mechanism for nanoplastics-induced lung injury through different lung cells, mouse inhalation models, and three-dimensional-based lung-on-a-chip, providing an insightful reference for pulmonary toxicity assessment of nanoplastics.

Advanced lung organoids and lung-on-a-chip for cancer research and drug evaluation: a review.

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.