Exploring effects of mechanical stress in the development of emphysema using a lung organoid

<bold>Background:</bold> Pathophysiology of emphysema in COPD is complex and still poorly understood. While tobacco exposure has long been implicated, recent insights suggest that mechanical stress at the alveolar level may also play a significant role in its development. Our study seeks to delve into the impact of mechanical stress and tobacco exposure on human alveolar epithelial cell (AEC) organoids, aiming to create functional alveolar model capable of mimicking emphysema through the manipulation of mechanical and biological factors, culminating in the development of 4D-alveolospheres. <bold>Material and methods:</bold> We subjected both lineage A549 cells and primary human AEC2 to a 2D-15% stretch at a rate of 15 cycles per minute ranging from 4 to 24 hours. Additionally, we stretched human lung alveolar organoids from AEC2s isolated by immunomagnetic sorting for 4 hours with similar intensity. Phenotypic analyses by qPCR and viability by flow cytometry analyses were performed at different time points (H0 to H24). <bold>Results:</bold> For A549 cells, viability remained above 90% even after 24 hours of stretch. qPCR analysis indicated a reduction in AEC1 markers (p2xr4 and pdpn), stability of intermediate cell markers (cldn4) and AEC2 markers (abca3). Likewise, viability of primary AEC2 cells was unaffected by 4 hours of stretch. Finally, 4 hours stretch applied to 3D organoids resulted in elastic deformation of the microwells while maintaining the integrity of organoids. <bold>Interpretation:</bold> We have successfully exposed human AEC2 to a mechanical stress simulating breathing in both 2D and 3D conditions paving the way for future experiments. Combining tobacco exposure and stretch will allow us to better replicate emphysema features.