A Modular Human Airway Lung‐Chip for Studying the Effect of Breathing‐Mechanics on Airway Epithelial Cell Biology

Abstract

Damage to the lung in respiratory disease involves complex interactions in the lung micro-environment. Such interactions involve different cell types (epithelial, mesenchymal, and immune cells) and mechanical forces (shear flow, pressure, and stretch). Traditional in vitro models, which are static and predominantly contain only one cell lineage, lack these interactions, limiting their physiological relevancy. Here, we present a new Airway Lung-Chip platform for studying airway epithelial cell biology in the context of mechanical stimulation and endothelial co-culture. We hypothesized that breathing-like stretch and directional airflow would modulate epithelial tissue thickness, cellular composition, and differentiation. To assess this hypothesis, we first developed a multi-step membrane functionalization method to overcome challenges hampering epithelial-endothelial co-cultures on stretchable, porous membranes at the air liquid interface. Such challenges included poor cell adhesion and undesired cellular migration between compartments. This optimized Airway Lung-Chip was then used to systematically explore the effect of biomechanical stretch mimicking the force and frequency of physiological breathing, as well as airflow, on the differentiation of primary human airway epithelial cells, in the presence or absence of an endothelial lining. Our preliminary results from probing genetic, cellular, and functional markers of epithelial cell types as well as histological sections of the tissue indicate that basal cells exposed to airflow and stretch differentiate towards a more small-airway like phenotype, containing few goblet cells and increased distribution of club cells, as compared to paired control chips. In conclusion, we have developed and optimized a new Airway Lung-Chip model that represents a more physiological model of the human airway for in vitro studies. Our data also indicate that biomechanical forces, such as stretch and directional airflow, can influence airway stem cell differentiation which may have important implications in developing models of ventilation or when studying mechanisms driving airway disease.