Engineering Decellularized Extracellular Matrix-Incorporated Apical-Out Airway Organoids.

The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial-ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization. Key features • This protocol incorporates dECM as size-refined microparticles, enabling a consistent and reproducible ECM input. • The workflow uses suspension culture to generate apicalout airway organoids, eliminating the need for matrix embedding while preserving ECM-cell interactions. • dECM-MPs are directly wrapped by epithelial cells, simplifying handling and supporting native-like epithelial-ECM integration. • The system is compatible with wholemount immunofluorescence staining for comprehensive epithelial lineage analysis.