Generation of Nasal Cell‐Derived Human Alveolar Organoids and Organoid‐Macrophage Assembloids for in Vitro Lung Modeling

We established robust protocols to generate physiological and functional alveolar organoids (nsoAlvO) from readily accessible and expandable nasal cell-derived organoids, and alveolar macrophages (monoAM) from peripheral blood monocytes. Through co-culture of nsoAlvO and monoAM, we generated organoid-macrophage assembloids, in which both components exhibited enhanced maturation. Comprehensive analyses, including immunostaining, functional assays, and single-cell RNA sequencing, demonstrated that the nsoAlvO and monoAM phenotypically and functionally resemble their native counterparts and engage in dynamic and extensive epithelial-macrophage communications. SSEA-1+ club cells were identified as the primary alveolar progenitor cells for nsoAlvO. Influenza virus infection in nsoAlvO revealed differential replicative fitness of H5N1 and H1N1 viruses, which recapitulate their authentic tropism in vivo. Notably, the addition of monoAM reduced H5N1 and H1N1 infection in nsoAlvO, suggesting a protective effect of alveolar macrophages against virus dissemination. These human alveolar organoids and organoid-macrophage assembloids provide universally accessible and physiologically relevant in vitro lung models for biomedical research and translational medicine.