Rapid expansion of primary human vocal fold epithelial cells via targeted pathway inhibition and anchorage-independent sphere culture.

Vocal fold epithelial cells (VFEs) serve critical physiologic and immunologic functions at the boundary between the upper and lower airways but are difficult to maintain and expand in primary cultures. This technical challenge has impeded progress in VFE biology as well as cell banking for translational applications. Here, using primary human VFEs, we show that simultaneous inhibition of transforming growth factor β (TGF-β), Rho-associated protein kinase (ROCK), and Notch signaling with a small-molecule inhibitor cocktail enables rapid proliferation, successful passaging, and long-term expansion while preserving the core epithelial phenotype. Under anchorage-independent culture conditions, VFE progenitors generate clonal spheres that can be expanded over multiple generations; sphere-dissociated VFEs then revert toward their original phenotype, which includes the ability to form stratified squamous epithelium in organotypic cocultures. Both pathway-inhibited and sphere-cultured VFEs exhibit mechanistically appropriate remodeling of the cellular proteome. These advances offer a robust toolkit for upper airway mucosal biology and regenerative medicine.