The past decade has witnessed tremendous endeavors to deliver novel preclinical in vitro lung models for pulmonary research endpoints, including foremost with the advent of organ- and lung-on-chips. With growing interest in aerosol transmission and infection of respiratory viruses within a host, most notably the SARS-CoV-2 virus amidst the global COVID-19 pandemic, the importance of crosstalk between the different lung regions (i.e., extra-thoracic, conductive and respiratory), with distinct cellular makeups and physiology, are acknowledged to play an important role in the progression of the disease from the initial onset of infection. In the present Methods article, we designed and fabricated to the best of our knowledge the first multi-compartment human airway-on-chip platform to serve as a preclinical in vitro benchmark underlining regional lung crosstalk for viral infection pathways. Combining microfabrication and 3D printing techniques, our platform mimics key elements of the respiratory system spanning (i) nasal passages that serve as the alleged origin of infections, (ii) the mid-bronchial airway region and (iii) the deep acinar region, distinct with alveolated airways. Crosstalk between the three components was exemplified in various assays. First, viral-load (including SARS-CoV-2) injected into the apical partition of the nasal compartment was detected in distal bronchial and acinar components upon applying physiological airflow across the connected compartment models. Secondly, nebulized viral-like dsRNA, poly I:C aerosols were administered to the nasal apical compartment, transmitted to downstream compartments via respiratory airflows and leading to an elevation in inflammatory cytokine levels secreted by distinct epithelial cells in each respective compartment. Overall, our assays establish an in vitro methodology that supports the hypothesis for viral-laden airflow mediated transmission through the respiratory system cellular landscape. With a keen eye for broader end user applications, we share detailed methodologies for fabricating, assembling, calibrating, and using our multi-compartment platform, including open-source fabrication files. Our platform serves as an early proof-of-concept that can be readily designed and adapted to specific preclinical pulmonary research endpoints.

Direct analysis of isolated mitochondria enables a better understanding of lung dysfunction. Despite well-defined mitochondrial isolation protocols applicable to other tissues, such as the brain, kidney, heart, and liver, a robust and reproductive protocol has not yet been advanced for the lung. We describe a protocol for the isolation of mitochondria from lung tissue aiming for functional analyses of mitochondrial O₂ consumption, transmembrane potential, reactive oxygen species (ROS) formation, ATP production, and swelling. We compared our protocol to that used for heart mitochondrial function that is well-established in the literature, and achieved similar results.