Endothelial cell stiffness and type drive the formation of biomechanically induced transcellular pores

Formation of transcellular pores facilitates material transport across endothelial barriers. In Schlemm's canal (SC) endothelium, impaired pore formation is linked to glaucoma, but the underlying processes remain poorly understood due to a lack of in vitro assays. Here, we present a platform to study pore formation in human endothelial cells. Pores were induced in SC cells by seeding them atop micron-sized magnetic beads and applying a magnetic field to generate basal-to-apical forces, mimicking in vivo biomechanics. Pore formation was dynamic, with pores opening and closing. Glaucomatous cells showed impaired pore formation, which correlated with increased stiffness. Notably, apical-to-basal forces failed to induce pores in SC cells but triggered pore formation in other endothelial types. These findings highlight the central role of cell mechanics in transcellular pore formation and provide an approach to investigate the mechanisms underlying this process.