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Ventilator-induced lung injury (VILI) is a potential threat to anyone receiving supportive mechanical ventilation for acute respiratory failure. Despite decades of research, however, the safest way to ventilate any given patient remains controversial. This makes fertile ground for novel concepts, and one that has arisen recently concerns the idea that a ventilator imparts potentially damaging mechanical energy to the lungs. The motivation for this concept is clear: energy transfer is involved when any structure becomes physically damaged. It may be intuitive, then, that the rate at which energy is delivered to the lungs by a ventilator, namely mechanical power, should be associated with VILI. Nevertheless, understanding the relationship between mechanical power and VILI requires clarity on the difference between stored versus dissipated energy regardless of whether ventilation is caused by positive pressure at the airway opening or negative pressure in the pleural space. 

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.