Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modeling

Submerged cultures of undifferentiated or transformed epithelial cells are widely used in respiratory research due to their ease of use and scalability. However, these systems fail to capture the cellular diversity of the human airway epithelium. Here, we describe a submerged differentiation model using cryopreserved human nasal epithelial cells obtained via minimally invasive brushings. By targeting Notch and BMP signaling with small molecule inhibitors, we differentiate these cells into complex epithelial cultures containing basal, secretory, and ciliated cell types on standard plastic cultureware. This method supports scalable culture of both 2D epithelial monolayers and 3D organoids and is applied to disease modeling in primary ciliary dyskinesia, cystic fibrosis, and respiratory syncytial virus infection. The resulting system enables scalable assessment of disease-relevant epithelial functions in respiratory research.