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.

外耳道 (EAC) 狭窄或闭锁通常需要皮肤移植来修复，但由于缺乏包含功能性腺体的移植物，感染和湿疹等术后并发症很常见。本研究的目的是通过分离和培养耵聍腺细胞来分离和表征用于构建含有耵聍腺的组织工程EAC皮肤的种子细胞。在这项研究中，EAC 皮肤样本是从成年山羊身上采集的，用于分离耵聍腺细胞。在不同传代评估细胞形态和增殖率、CK7、CK8、CK18 和 CK19（腺细胞特异性标志物）的表达以及 β-防御素-1、溶菌酶和多糖的分泌，以验证耵聍腺细胞的存在并确定功能和增殖潜力是否得以维持。成功地从山羊 EAC 皮肤中分离和提取了耵聍腺。此外，分离的腺细胞保持了强大的增殖潜力，在该培养系统中表现出 CK7、CK8、CK18 和 CK19 的高表达，并大量分泌 β-防御素-1、溶菌酶和多糖。然而，随着传代次数的增加，腺细胞特异性标志物的表达和分泌功能逐渐下降，表明传代培养的耵聍腺细胞在传代5次后去分化。总之，使用含血清培养系统成功地从山羊EAC皮肤中分离、培养和扩增了耵聍腺。重要的是，分离的腺细胞保留了强大的增殖潜力，并在早期传代（P1-P3）中保持了它们的表型和功能，