Recapitulating apicobasal tissue polarity in extracellular matrix-incorporated airway organoids.

The airway epithelium is a dynamic barrier that interfaces with the external environment and internal matrix along its apicobasal axis. To recapitulate this spatial organization and associated cell-ECM interactions in an organoid format, we present the decellularized extracellular matrix-incorporated Apical-out Airway Organoid (dECM-AoAO), which integrates basolateral matrix cues through the incorporation of human lung dECM microparticles (dECM-MPs), while maintaining direct apical exposure to the exterior. Compared to ECM-free AoAOs, dECM incorporation diversifies lineage distribution, more closely recapitulating native epithelial composition and responsiveness to pathogenic stimulation. Harnessing dECM-AoAO locomotion driven by outward-facing ciliary beating, we developed an experimental and computational pipeline for batch analysis of organoid motility as a functional readout of ciliary activity. Furthermore, dECM-AoAOs are compatible with cryopreservation, retaining viability, lineage composition, and ciliary function upon revival. Together, this work establishes the dECM-AoAO as a physiologically relevant model system for investigating epithelial-ECM crosstalk during airway homeostasis, pathogenesis, and injury responses.