Droplet microfluidics-enabled large-scale generation of apical-out human nasal organoids for respiratory virus infection modeling

Human nasal organoids (HNOs) have emerged as promising human-derived models for respiratory virus studies by recapitulating the cellular diversity and infection-related responses of the nasal epithelium. However, scalable generation of HNOs remains technically challenging, limiting their broader application in systematic respiratory virus infection studies and antiviral evaluation. Here, we developed a droplet microfluidics-based platform for the large-scale generation of apical-out HNOs using cell-laden microgels produced from uniform droplets. The platform continuously generated approximately 90,000–120,000 cell-laden droplets per minute, supporting efficient organoid formation with an average efficiency of 71%. The resulting HNOs exhibited apical-out polarity and contained major nasal epithelial cell types. Using human rhinovirus C15 (RV-C15) as a model respiratory virus, we demonstrated that these apical-out HNOs supported productive RV-C15 replication and recapitulated key infection-associated phenotypes, including cell death, innate immune responses, and ciliary damage. Furthermore, these HNOs enabled antiviral drug evaluation, with rupintrivir and itraconazole reducing RV-C15 replication and virus-induced cytotoxicity. We established a droplet microfluidics-based platform for the large-scale generation of apical-out HNOs. By integrating controlled microgel generation, reproducible organoid formation, and direct apical accessibility, this platform provides a scalable and human-relevant approach for respiratory virus infection modeling and antiviral evaluation. Not applicable.