Isolation, culture, and identification of ceruminous gland cells

External auditory canal (EAC) stenosis or atresia usually requires a skin graft to repair, but due to the lack of a graft containing functional glands, postoperative complications such as infection and eczema are common. The aim of this study was to isolate and characterize seed cells for the construction of tissue engineered EAC skin containing ceruminous gland by isolating and cultivating cells of ceruminous gland. In this study, EAC skin samples were harvested from adult goats for ceruminous gland cell isolation. Cell morphology and proliferation rates, expression of CK7, CK8, CK18, and CK19 (glandular cell specific-markers), and secretion of β-defensin-1, lysozyme, and polysaccharides were evaluated at different passages to verify the presence of ceruminous gland cells and determine whether function and proliferation potential were maintained. Ceruminous glands were successfully isolated and extracted from goat EAC skin. Furthermore, the isolated glandular cells maintained robust proliferation potential, exhibited high expression of CK7, CK8, CK18, and CK19, and vigorously secreted β-defensin-1, lysozyme, and polysaccharides in this culture system. However, expression of glandular cell specific-markers and secretory function gradually declined with increasing passage number, indicating dedifferentiation of the subcultured ceruminous gland cells after five passages. In conclusion, ceruminous glands were successfully isolated, cultured, and expanded from goat EAC skin using the serumcontaining culture system. Importantly, the isolated glandular cells retained robust proliferation potential and maintained their phenotype and function in early passages (P1-P3), indicating the method's potential application for ceruminous gland regeneration.