Human nasal organoids sustain reproducible airway epithelial differentiation and influenza virus-host innate immune interaction

The cultures of human nasal epithelial cells (NECs) are good surrogate models for studying immune responses. Three-dimensional (3D) organoids have emerged as an alternative for nasal epithelium, serving as robust platforms for modeling respiratory viral infections. We aimed to generate human NEC organoids and to determine their cellular characteristics as an in vitro model for air-liquid interface (ALI) culture and influenza A virus (IAV) infection. Primary human nasal mucosal tissues were obtained from healthy donors undergoing nasal surgery, and nasal epithelial cell (NEC) organoids were established using Matrigel-based three-dimensional culture. Differentiated NEC organoids and air–liquid interface (ALI) cultures were characterized by histology, immunostaining, electron microscopy, and quantitative real-time PCR, followed by influenza A virus (A/WS/33, H1N1) infection assays. In addition, single-cell RNA sequencing (scRNA-seq) was performed to analyze cellular heterogeneity and differentiation trajectories of NEC organoids. We successfully developed human NEC organoids with using a Matrigel-based 3D culture system that recapitulates the structural characteristics and cellular composition of the human nasal epithelium. Histological results, real-time PCR, and single-cell RNA sequencing (scRNA-seq) revealed that the organoids consisted of seven different cell types and displayed a difference in the composition between the cells. The organoids displayed a much higher rate of basal progenitor cells whereas ciliated cells were less dominant. Unlike ALI cultures in which passage (P) 2 was the limit, the organoids exhibited successful and subsequent passages up to P5. Our data determined that NEC organoids are an adequate in vitro model for IAV infection and showed a sharp induction of mRNA levels for interferons (IFNs) and IFN-stimulated genes following IAV infection. This study’s results demonstrate that human NEC organoids serve as a robust in vitro model, successfully recapitulating the biological characteristics of nasal epithelium, and could be an adequate tool for exploring distinct IFN-related innate immune responses following influenza infection.