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Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Here, we present a reliable, and simply reproducible process for constructing user-controlled long rounded extracellular matrix (ECM)-embedded vascular microlumens on-chip for endothelization and co-culture with stromal cells obtained from human lung. We demonstrate the critical impact of microchannel cross-sectional geometry and length on uniform distribution and magnitude of vascular wall shear stress, which is key when emulating in vivo-observed blood flow biomechanics in health and disease. In addition, we provide an optimization protocol for multicellular culture and functional validation of the system. Moreover, we show the ability to finely tune rheology of the three-dimensional natural matrix surrounding the vascular microchannel to match pathophysiological stiffness. In summary, we provide the scientific community with a matrix-embedded microvasculature on-chip populated with all-primary human-derived pulmonary endothelial cells and fibroblasts to recapitulate and interrogate lung parenchymal biology, physiological responses, vascular biomechanics, and disease biogenesis in vitro. Such a mix-and-match synthetic platform can be feasibly adapted to study blood vessels, matrix, and ECM-embedded cells in other organs and be cellularized with additional stromal cells.
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Electronic cigarette (e-cigarette) use continues to rise globally. E-cigarettes have been presented as safer alternatives to combustion cigarettes that can mitigate the harm associated with tobacco products; however, the degree to which e-cigarette use itself can lead to morbidity and mortality is not fully defined. Herein we describe how e-cigarettes function; discuss the current knowledge of the effects of e-cigarette aerosol on lung cell cytotoxicity, inflammation, antipathogen immune response, mucociliary clearance, oxidative stress, DNA damage, carcinogenesis, matrix remodelling and airway hyperresponsiveness; and summarise the impact on lung diseases, including COPD, respiratory infection, lung cancer and asthma. We highlight how the inclusion of nicotine or flavouring compounds in e-liquids can impact lung toxicity. Finally, we consider the paradox of the safer cigarette: the toxicities of e-cigarettes that can mitigate their potential to serve as a harm reduction tool in the fight against traditional cigarettes, and we summarise the research needed in this underinvestigated area.
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Sistemas Eletrônicos de Liberação de Nicotina , Pulmão , Humanos , Pulmão/efeitos dos fármacos , Pneumopatias/induzido quimicamente , Nicotina/efeitos adversos , Redução do Dano , Estresse Oxidativo , Vaping/efeitos adversos , Dano ao DNA , Produtos do Tabaco/efeitos adversosRESUMO
Flavored electronic cigarettes (ECs) present a serious health challenge globally. Currently, it is unknown whether the addition of highly popular menthol flavoring to e-liquid is associated with changes in the number of aerosolized particles generated or altered lung function. Here, we first performed preclinical studies using our novel robotic platform Human Vaping Mimetic Real-Time Particle Analyzer (HUMITIPAA). HUMITIPAA generates fresh aerosols for any desired EC in a very controlled and user-definable manner and utilizes an optical sensing system to quantitate and analyze sub-micron and microparticles from every puff over the course of vaping session in real-time while emulating clinically relevant breathing mechanics and vaping topography. We discovered that addition of menthol flavoring to freshly prepared e-liquid base propylene glycol-vegetable glycerin leads to enhanced particle counts in all tested size fractions, similar to the effect of adding vitamin E acetate to e-liquid we previously reported. Similarly, we found that menthol vs. non-menthol (tobacco) flavored pods from commercially available ECs leads to generation of significantly higher quantities of 1-10 µm particles upon inhalation. We then retrospectively analyzed data from the COPDGene study and identified an association between the use of menthol flavored ECs and reduced FEV1% predicted and FEV1/FVC independent of age, gender, race, pack-years of smoking, and use of nicotine or cannabis-containing vaping products. Our results reveal an association between enhanced inhaled particle due to menthol addition to ECs and worse lung function indices. Detailed causal relation remains to be demonstrated in future large-scale prospective clinical studies. Importantly, here we demonstrate utility of the HUMITIPAA as a predictive enabling technology to identify inhalation toxicological potential of emerging ECs as the chemical formulation of e-liquid gets modified.
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Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Humanos , Fumantes , Mentol/efeitos adversos , Estudos Prospectivos , Estudos Retrospectivos , Produtos do Tabaco/efeitos adversos , PulmãoRESUMO
The human lung is constantly exposed to the environment and potential pathogens. As the interface between host and environment, the respiratory epithelium has evolved sophisticated sensing mechanisms as part of its defense against pathogens. In this review, we examine how the respiratory epithelium senses and responds to influenza A virus, the biggest cause of respiratory viral deaths worldwide.
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Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Humanos , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/virologiaRESUMO
Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.
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Epitélio/efeitos dos fármacos , Inflamação/metabolismo , Interleucina-13/farmacologia , Dispositivos Lab-On-A-Chip , Pneumopatias/tratamento farmacológico , Pneumopatias/metabolismo , Humanos , Inflamação/patologia , Técnicas de Cultura de TecidosRESUMO
4-1BB is expressed on invariant (i)NKT cells, but its role is unclear. We showed previously that iNKT cells are involved in control of monocyte numbers during influenza A virus (IAV) infection and now question the role of the 4-1BB costimulatory pathway in the cross-talk between these cells. We found that iNKT cells and monocytes interact to promote expression of 4-1BB and 4-1BBL, respectively. Blockade of 4-1BB/L pathway under resting coculture conditions increased apoptosis of iNKT cells and monocytes. However, activation of iNKT cells overrides this survival signal, causing marked apoptosis of monocytes independent of 4-1BB/L. Blocking 4-1BBL in alpha-galactosylceramide-activated iNKT-monocyte cocultures reduced iNKT proliferation and abrogated monocytic IL-12 production. In vivo, expression of 4-1BB and 4-1BBL is increased on iNKT cells and Ly6C(hi) monocytes, respectively, during IAV infection, and there were lower frequencies of apoptosing Ly6C(hi) monocytes in the blood of iNKT knockout mice and higher numbers of monocytes in lungs compared with infected wild-type mice. Adoptive transfer of iNKT cells into the lungs of these mice reduced lung Ly6C(hi) monocytes levels, even when iNKT cells were preincubated with 4-1BB blocking Abs. These findings suggest that under resting conditions, 4-1BB/L engagement during iNKT-monocyte interaction promotes survival of these cells. When iNKT cells are activated, whether by alpha-galactosylceramide or during IAV infection, iNKT cells induced apoptosis of monocytes via a 4-1BB/L-independent mechanism, reducing monocyte numbers. 4-1BB/L costimulation amplified monocyte-mediated proliferation of iNKT cells, indirectly providing a method for monocytes to control their own numbers during infection.
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Ligante 4-1BB/metabolismo , Monócitos/metabolismo , Células T Matadoras Naturais/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose/imunologia , Comunicação Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Técnicas de Cocultura , Citocinas/biossíntese , Humanos , Vírus da Influenza A/imunologia , Ativação Linfocitária/imunologia , Camundongos , Monócitos/imunologia , Células T Matadoras Naturais/imunologiaAssuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Pneumologia/métodos , Animais , Modelos Animais de Doenças , Humanos , Técnicas In Vitro/instrumentação , Técnicas In Vitro/métodos , Camundongos , Doença Pulmonar Obstrutiva Crônica/terapiaRESUMO
Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Lung vasculature is particularly important due to its involvement in genesis and progression of rare, debilitating disorders as well as common chronic pathologies. Here, we provide an overview of the latest advances in the development of pulmonary vascular (PV) models using emerging microfluidic tissue engineering technology Organs-on-Chips (so-called PV-Chips). We first review the currently reported PV-Chip systems and their key features, and then critically discuss their major limitations in reproducing in vivo-seen and disease-relevant cellularity, localization, and microstructure. We conclude by presenting latest efforts to overcome such technical and biological limitations and future directions.
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Modelos Biológicos , Engenharia Tecidual , Humanos , MicrofluídicaRESUMO
"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.
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Tecnologia Biomédica , Pneumologia , Ciência Translacional Biomédica , Humanos , Estados UnidosRESUMO
Individuals with Down syndrome (DS) clinically manifest severe respiratory illnesses; however, there is a paucity of data on how DS influences homeostatic physiology of lung airway, and its reactive responses to pulmonary pathogens. We generated well-differentiated ciliated airway epithelia using tracheas from wild-type and Dp(16)1/Yey mice in vitro, and discovered that Dp(16)1/Yey epithelia have significantly lower abundance of ciliated cells, an altered ciliary beating profile, and reduced mucociliary transport. Interestingly, both sets of differentiated epithelia released similar quantities of viral particles after infection with influenza A virus (IAV). However, RNA-sequencing and proteomic analyses revealed an immune hyperreactive phenotype particularly for monocyte-recruiting chemokines in Dp(16)1/Yey epithelia. Importantly, when we challenged mice in vivo with IAV, we observed immune hyper-responsiveness in Dp(16)1/Yey mice, evidenced by higher quantities of lung airway infiltrated monocytes, and elevated levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Our findings illuminate mechanisms underlying DS-mediated pathophysiological changes in airway epithelium.
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We recently developed a robotic human vaping mimetic real-time particle analyzer (HUMITIPAA) to evaluate the impact of change in chemical constituents and breathing profiles of electronic cigarettes (ECs) on potential pulmonary toxicity. Here, we describe the fabrication procedure of EC mouthpiece(s), establishment of sensor saturation curve, and preparation of e-liquid and vaping device(s) for testing. We further detail steps for HUMITIPAA preparation and connection setup, followed by data collection and processing. For complete details on the use and execution of this protocol, please refer to Kaiser et al. (2021).1.
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Sistemas Eletrônicos de Liberação de Nicotina , Robótica , Vaping , Humanos , Vaping/efeitos adversos , Biomimética , Coleta de DadosRESUMO
Responding quickly to emerging respiratory viruses, such as SARS-CoV-2 the causative agent of coronavirus disease 2019 (COVID-19) pandemic, is essential to stop uncontrolled spread of these pathogens and mitigate their socio-economic impact globally. This can be achieved through drug repurposing, which tackles inherent time- and resource-consuming processes associated with conventional drug discovery and development. In this review, we examine key preclinical and clinical therapeutic and prophylactic approaches that have been applied for treatment of SARS-CoV-2 infection. We break these strategies down into virus- versus host-targeting and discuss their reported efficacy, advantages, and disadvantages. Importantly, we highlight emerging evidence on application of host serine protease-inhibiting anticoagulants, such as nafamostat mesylate, as a potentially powerful therapy to inhibit virus activation and offer cross-protection against multiple strains of coronavirus, lower inflammatory response independent of its antiviral effect, and modulate clotting problems seen in COVID-19 pneumonia.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , PandemiasRESUMO
In vitro culture and differentiation of human-derived airway basal cells under air-liquid interface (ALI) into a pseudostratified mucociliated mucosal barrier has proven to be a powerful preclinical tool to study pathophysiology of respiratory epithelium. As such, identifying differentiation stage-specific biomarkers can help investigators better characterize, standardize, and validate populations of regenerating epithelial cells prior to experimentation. Here, we applied longitudinal transcriptomic analysis and observed that the pattern and the magnitude of OMG, KRT14, STC1, BPIFA1, PLA2G7, TXNIP, S100A7 expression create a unique biosignature that robustly indicates the stage of epithelial cell differentiation. We then validated our findings by quantitative hemi-nested real-time PCR from in vitro cultures sourced from multiple donors. In addition, we demonstrated that at protein-level secretion of BPIFA1 accurately reflects the gene expression profile, with very low quantities present at the time of ALI induction but escalating levels were detectable as the epithelial cells terminally differentiated. Moreover, we observed that increase in BPIFA1 secretion closely correlates with emergence of secretory cells and an anti-inflammatory phenotype as airway epithelial cells undergo mucociliary differentiation under air-liquid interface in vitro.
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Células Epiteliais , Mucosa Respiratória , Humanos , Células Cultivadas , Células Epiteliais/metabolismo , Diferenciação Celular , Epitélio , Biomarcadores/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismoRESUMO
Antiviral strategies that target host systems needed for SARS-CoV-2 replication and pathogenesis may have therapeutic potential and help mitigate resistance development. Here, we evaluate nafamostat mesylate, a potent broad-spectrum serine protease inhibitor that blocks host protease activation of the viral spike protein. SARS-CoV-2 is used to infect human polarized mucociliated primary bronchiolar epithelia reconstituted with cells derived from healthy donors, smokers and subjects with chronic obstructive pulmonary disease. Nafamostat markedly inhibits apical shedding of SARS-CoV-2 from all donors (log10 reduction). We also observe, for the first-time, anti-inflammatory effects of nafamostat on airway epithelia independent of its antiviral effects, suggesting a dual therapeutic advantage in the treatment of COVID-19. Nafamostat also exhibits antiviral properties against the seasonal human coronaviruses 229E and NL6. These findings suggest therapeutic promise for nafamostat in treating SARS-CoV-2 and other human coronaviruses.
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Vitamin E acetate (VEA) has been strongly linked to outbreak of electronic cigarette (EC) or vaping product use-associated lung injury. How VEA leads to such an unexpected morbidity and mortality is currently unknown. To understand whether VEA impacts the disposition profile of inhaled particles, we created a biologically inspired robotic system that quantitatively analyzes submicron and microparticles generated from ECs in real-time while mimicking clinically relevant breathing and vaping topography exactly as happens in humans. We observed addition of even small quantities of VEA was sufficient to alter size distribution and significantly enhance total particles inhaled from ECs. Moreover, we demonstrated utility of our biomimetic robot for studying influence of nicotine and breathing profiles from obstructive and restrictive lung disorders. We anticipate our system will serve as a novel preclinical scientific research, decision-support tool when insight into toxicological impact of modifications in electronic nicotine delivery systems is desired.
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The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.
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Antivirais/farmacologia , Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , Teste para COVID-19/métodos , Dispositivos Lab-On-A-Chip , Animais , COVID-19/diagnóstico , COVID-19/virologia , Linhagem Celular , Cricetinae , Feminino , Proteínas de Fluorescência Verde , Humanos , Masculino , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
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.
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Biomimética/instrumentação , Exposição Ambiental/efeitos adversos , Inalação , Dispositivos Lab-On-A-Chip , Robótica , Fumar/efeitos adversos , Linhagem Celular , HumanosRESUMO
Lung disorders such as chronic obstructive pulmonary disease (COPD) and lower respiratory tract infections (LRTIs) are leading causes of death in humans globally. Cigarette smoking is the principal risk factor for the development of COPD, and LRTIs are caused by inhaling respiratory pathogens. Thus, a thorough understanding of host-environment/pathogen interactions is crucial to developing effective preventive and therapeutic modalities against these disorders. While animal models of human pulmonary conditions have been widely utilized, they suffer major drawbacks due to inter-species differences, hindering clinical translation. Here we summarize recent advances in generating complex 3D culture systems that emulate the microarchitecture and pathophysiology of the human lung, and how these platforms have been implemented for studying exposure to environmental factors, airborne pathogens, and therapeutic agents.
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The Health Law, Policy & Ethics Project at Emory University School of Law and the Human Toxicology Project Consortium of the Humane Society of the United States co-sponsored a symposium on October 23, 2017, to showcase innovations using human-based in silico and in vitro models for drug and device discovery. The goal of the symposium was to introduce researchers and students to exciting new tools and possible future careers that will increase understanding of disease and improve the search for effective therapeutics, while reducing reliance on animal testing. The symposium concluded with a discussion between scientists and lawyers about the legal regulation of new biomedical research technologies.
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Pesquisa Biomédica/legislação & jurisprudência , Pesquisadores/legislação & jurisprudência , Tecnologia Farmacêutica/legislação & jurisprudência , Animais , Humanos , Estados UnidosRESUMO
Respiratory tissues are bombarded by billions of particles daily. If allowed to accumulate, these particles can cause injury, inflammation, or infection, and thus may significantly disrupt airflow and gas exchange. Mucociliary defense, a primary mechanism for protecting host tissues, operates through the coordinated functions of mucus and cilia that trap and eliminate inhaled materials. Mucociliary function is also required for the elimination of endogenous cells and debris. Although defense is necessarily robust, it is also tightly regulated to minimize physiologic disruption of the host. Indeed, mucociliary dysfunction contributes to the pathogenesis of many lung diseases-including asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis-in which airflow limitation, inflammation, persistent tissue injury, and structural remodeling occur. Here, we highlight recent advances in cilia and mucin biology, the importance of well-controlled mucociliary interactions, and the need to better understand how these regulate innate barrier and immune defense.