Deciphering emphysema pathophysiology through an engineered 3D-lung organoid

<bold>Background:</bold> Three-dimensional (3D) models of emphysema, provide a unique tool to improve knowledge in underlying involved pathways. The biggest challenge remains to reduce the heterogeneity of alveolospheres which are currently mostly grown in Matrigel. Our aim is to study emphysema pathways using an adjustable, sustainable and reproductible 3D-alveolosphere model from human type II alveolar epithelial cell (AEC2). <bold>Methods:</bold> AEC2 cells isolated by immunomagnetic sorting (HTII-280+) from 39 lung samples of smoker and non-smoker patients were cultured in hydrogel microwells of adjustable shape, stiffness and size by photopolymerisation. Morphological and phenotypic analyses were performed at D1, 7, 14 and 21 (immunolabelling, qPCR, electron microscopy (TEM)). Emphysema, was modelled through exposure to 5% cigarette smoke extract (CSE) during 5 consecutive days (qPCR, calcein, TEM). <bold>Results:</bold> Progressive formation of a central lumen was observed from D7 and maintained till D21. AEC1 markers (p2xr4, pdpn expression) appears progressively while AEC2 markers persisted over time (abca3, sftpa, sftpc expression). Tight junctions suggest epithelial barrier formation (TEM, ZO-1 immunostaining) and lamellar bodies, surfactant synthesis (TEM). CSE exposure led to a decrease in cell viability, architectural disorganisation, increase in oxidative stress markers (hmox, nqo1, srxn1 expression) and release of cytokines (MIF, IL8) in the supernatant. <bold>Interpretation:</bold> An adjustable and reproducible 3D-alveolosphere model from isolated human AEC2 was obtained reproducing the main characteristics of in vivo alveoli. Exposure to CSE provides an opportunity to study the pathophysiological pathways involved in emphysema.