The impact of mechanical stresses induced by flow and stretch on barrier function in airway epithelial cells

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Background: Much of the basic biology known about the airway epithelium has been deduced from static in vitro cell culture systems. Our understanding of airway epithelial cell biology may therefore be limited and under appreciate the impact of dynamic forces on biological properties important in epithelial barrier function. Our overarching objective is to incorporate physiological ranges of flow and stretch into our in vitro airway epithelial cell culture models and explore barrier function properties. ******Incorporation of flow into in vitro cell culture systems has showed that baseline mucus secretion is much higher than previously considered. Furthermore, incorporation of compressive stress showed that airway epithelial cells move within a monolayer and are not fixed in position. Incorporating flow and stretch into in vitro cell culture models of airway epithelial cells is likely to reveal unexplored biology. ******Aims: Use in vitro cell culture models to explore the impact of i) a physiological range of flows, ii) a physiological range of stretch, and iii) a combination of flow and stretch on airway epithelial cell barrier function******Methods: All three aims have experiments with outcome measurements that explore epithelial barrier function measured by transepithelial electrical resistance (TEER), molecular transport, wound repair, and gene expression. ******We will use a human airway epithelial cell line, Calu-3, to perform our experiments as this cell line creates tight mechanical barriers and has been validated for molecular transport studies. Experiments will be performed on cells grown under submerged monolayer and, where possible, air-liquid interface culture conditions. We will combine our cell cultures with a polydimethylsiloxane (PDMS) organ-on-a-chip device to allow integration of perfusion and stretch. For perfusion experiments, a syringe pump will be used to expose cells to a range of flow rates based on in vivo resting and peak flow rates. For stretch experiments, an adapted syringe pump system with an occlusion valve will be used to induce pressure changes that are transduced in vivo in response to stretch. To explore the potential for interactions between flow and stretch, we will combine these mechanical forces on our PDMS device.******Significance: Nascent work by others and our preliminary data suggest that mechanical forces induced by flow and stretch impact airway epithelial cell biology. Our proposed research program will explore unknown areas of airway epithelial cell biology by integrating physiological conditions currently absent from conventional cell culture systems, creating a foundation of knowledge for future basic and health research discoveries. Furthermore, our program will develop and validate technology and methods, protected by intellectual property, that can be adapted to other cell types to study how tissue specific mechanical forces impact function.