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.
Impact Statement
In this study, we developed a convenient and reproducible method for long-term culture of murine nasopharyngeal epithelial cell (NEC) lines with NEC medium. NEC medium continuously maintained primary murine NECs with high proliferating activity, selectively supported the growth of NECs rather than fibroblasts and mesenchymal cells, and maintained NECs not undergoing epithelial–mesenchymal transition and being tumorigenic. This study provides a valuable cellular tool for investigating nasopharyngeal epithelial-related diseases such as nasopharyngeal carcinoma and respiratory virus infection.
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