Bidirectional airflow in lung airway-on-a-chip with matrix-derived membrane elicits epithelial glycocalyx formation

Organ-on-a-chip systems are rapidly advancing as a viable alternative to existing experimental models in respiratory research. To date, however, epithelial cell cultures within lung airway-on-a-chip devices have yet to demonstrate the presence of an epithelial glycocalyx, a thin layer of proteoglycans, glycoproteins, and glycolipids known to play an important role in regulating epithelial function. Here, we demonstrate that an airway-on-a-chip device that incorporates bidirectional flow mimicking breathing cycles in combination with an ultra-thin matrix-derived membrane (UMM) layer can generate a glycocalyx layer comprised of heparan sulfate. Results with this device and airflow system showed dramatic differences of airway epithelial cell viability and expression of tight junctions, cilia, and mucus over a wide range of flow rates when cultured under oscillatory flow. More importantly, for the first time in a microfluidic organ-on-a-chip setting, we achieved the visualization of an airflow-induced epithelial glycocalyx layer. Our experiments highlight the importance of physiological mimicry in developing in vitro models, as bidirectional airflow showed more representative mucociliary differentiation compared to continuous unidirectional airflow. Thus, the lung airway-on-a-chip platform demonstrated in this study holds great potential as a lung epithelial barrier model for studying the mechanisms of various respiratory diseases and for testing the efficacy of therapeutic candidates in the presence of bidirectional airflow and the glycocalyx. A unique lung airway-on-a-chip device combines an ultra-thin matrix-derived membrane with bidirectional airflow over a confluent airway epithelium, leading to presence of epithelial glycocalyx.