A novel alveoli-on-chip platform for modeling cyclic stretch in patient-derived alveolar epithelial cells cultured from organoids

We report a novel alveoli-on-chip (AOC) platform that replicates an array of alveoli closely matching <i>in vivo</i> dimensions and enables three-dimensional cyclic stretching to simulate the respiratory movements experienced by the alveolar epithelium. This system is based on an arrangement of parallel pneumatic channels with a sinusoidal wall morphology leading to interconnected circular chambers with a 250 μm radius. Above the pneumatic channels is a thin flexible membrane on which alveolar epithelial cells are cultured. When pressure is applied, the membrane deforms, with maximum deformation in the centre of these chambers. To simplify cell culture management, the AOC has an open design and is configured for medium throughput in a 24-well plate format. The chip is populated with patient-derived lung alveolar epithelial cells from organoids. Type 2 lung alveolar epithelial cells (AT2) were magnetically sorted from tissue samples obtained from patients undergoing lung surgery and cultured as organoids for expansion. The expansion efficiency of AT2 patient-derived organoids was optimized by the supplementation of endothelial supplied alveolar potentiators (ESAP), resulting in a 2.5-fold increase in organoid size increase after 10 days of culture compared to without ESAP. Immunostaining of expanded organoids confirmed their epithelial identity (EpCAM⁺) and demonstrated the presence of AT2s (SFTPC⁺, HTII-280⁺), AT1s (PDPN⁺), and transitional AT0s (KRT8⁺) subtypes. Dissociated lung organoids cells from three patients were cultured on the chip under conditions with or without physiological mechanical stress. Bulk RNA sequencing revealed that cyclic stretch significantly influences gene expression in lung alveolar epithelial cells. An increase in the expression of genes associated with autophagy, G2/M checkpoint and mTORC1 was observed, indicating cellular stress responses and adaptation to mechanical stimuli. Thus, this study not only showcases the AOC's potential to recapitulate alveolar epithelium mechanical cues but also offers a robust and scalable platform to be used for research and preclinical applications.