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

## TL;DR

Researchers developed a method to rapidly grow human vocal fold epithelial cells in the lab using drug combinations and special culture techniques, which could help advance vocal fold research and regenerative medicine.

## Contribution

A novel small-molecule inhibitor cocktail and anchorage-independent culture method enable long-term expansion of primary human vocal fold epithelial cells.

## Key findings

- Inhibiting TGF-β, ROCK, and Notch signaling allows rapid proliferation and long-term expansion of vocal fold epithelial cells.
- Anchorage-independent culture promotes the formation of clonal epithelial spheres from progenitor cells.
- Both methods alter the cellular proteome in ways consistent with their biological mechanisms.

## Abstract

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.

•TGF-β, ROCK, and Notch inhibition drive vocal fold epithelial cell proliferation•Anchorage-independent culture promotes formation of clonal epithelial spheres•Pathway inhibition and sphere culture differentially alter the cellular proteome

TGF-β, ROCK, and Notch inhibition drive vocal fold epithelial cell proliferation

Anchorage-independent culture promotes formation of clonal epithelial spheres

Pathway inhibition and sphere culture differentially alter the cellular proteome

Primary vocal fold epithelial cells are challenging to culture and expand in vitro, hampering progress in vocal fold biology and its translational applications. To address the technical challenges of limited proliferation and growth arrest, we identified and tested a small-molecule inhibitor cocktail targeting TGF-β, ROCK, and Notch signaling pathways and introduced an anchorage-independent culture technique to facilitate formation of progenitor-enriched clonal spheres.

Shi et al. report improved primary culture of human vocal fold epithelial cells by using a small-molecule inhibitor cocktail that targets TGF-β, ROCK, and Notch signaling and by employing a free-floating sphere-formation technique. These complementary methods enable rapid population expansion and progenitor enrichment for research and translational applications.