Modelling cigarette smoke-induced lung vascular dysfunction using an alveolus-on-chip

Abstract The alveolus is central to gas exchange in the lung, and alveolar damage is a characteristics of a variety of lung diseases. Understanding alveolar microvascular dynamics and epithelial-endothelial interactions is essential for accurately modeling alveolar physiology and its dysfunction in lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis and acute respiratory distress syndrome (ARDS). In this study, we present an open-top, membrane-free alveolus-on-chip platform incorporating self-assembled, perfusable 3D vascular networks by primary human lung endothelial cells and pericytes, co-cultured with alveolar epithelial type 2 (AEC2) cells. These vascular networks were developed within 6 days under continuous flow and remained stable for at least 12 days. The inclusion of pericytes supported capillary-like vessel formation and increased gene expression of EDNRB1 , a gene enriched in alveolar microvascular endothelial cells. Furthermore, CD31 gene expression was higher in 3D endothelial networks compared to 2D endothelial monolayers, suggesting increased cell-cell adhesion. Monocytes could be successfully perfused through the networks, expanding the platform’s potential for studying immune interactions in lung disorders. Culturing AEC2 monolayers directly on the vascularized hydrogel enabled physiologically relevant cell-cell interactions without artificial membranes, while maintaining air-liquid interface conditions. Importantly, exposure of the AEC2 layer to whole cigarette smoke (WCS) led to complete disintegration of the underlying vascular network, an effect not observed in the absence of AEC2. This chip model provides a human-relevant system for investigating vascular-epithelial crosstalk in the alveolus, smoke-induced lung injury, and immune recruitment, offering a valuable platform for future disease modeling and drug testing applications.