Establishment of Long-Term Expansion Culture for Primary Murine Nasopharyngeal Cells

The nasopharynx constitutes a critical component of the respiratory tract. Nasopharyngeal diseases are closely related to nasopharyngeal epithelial cells (NECs). Considering the current paucity of appropriate cell models for studying nasopharynx-related diseases, there is an urgent need to develop a simple and efficient method for the long-term culture and robust expansion of primary NECs. In this study, we employed the NEC medium supplemented with Wnt3a, R-spondin, Noggin, and other growth factors to stimulate the proliferation of nasopharyngeal epithelial stem cells and maintain their self-renewal state, enabling long-term culture. Leveraging this strategy, we successfully developed a simplified and efficient method for long-term culture of primary murine NECs. The NEC medium provided a selective advantage for stably expanding cytokeratin 5- and epithelial membrane antigen-positive epithelial cells rather than alpha-smooth muscle actin-marked fibroblasts and prevented epithelial-mesenchymal transition as evidenced by continuously strong E-cadherin expression and being negative for vimentin. The established NEC line exhibited stable long-term proliferation with no evident signs of senescence. We also confirmed the nontumorigenic nature of the established nasopharyngeal cell line in mice. Our findings from this study provided a valuable cellular tool for investigating nasopharyngeal epithelial-related diseases and developing therapeutic strategies.