Amesara R, Kim Y, Sano S, Harada T, Juhn S K
Department of Otolaryngology, University of Minnesota Medical School, Minneapolis.
Eur Arch Otorhinolaryngol. 1992;249(3):164-7. doi: 10.1007/BF00183493.
A reproducible method is presented for primary cultures of middle ear epithelial cells (MEEC) from chinchillas. The MEEC were first dissociated with protease and grown on collagen-coated membrane using a culture medium containing equal volumes of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 0.5% fetal bovine serum. Outgrowth of cells was first noted within 24 h, reaching confluency in 6-7 days. These cells grew in a monolayer and appeared to be ovoid or polygonal. By immunofluorescence microscopy, these cells stained for cytokeratin, but not for type III collagen. In contrast, fibroblasts stained for type III collagen, but not for cytokeratin. Based on growth characteristics, morphology, and immunofluorescent findings, these cells were determined to be epithelial cells. To retard the outgrowth of fibroblasts, 5 mM putrescine was added to the culture medium on the 2nd day of explant. Contamination with fibroblasts was consistently less than 5% when defined as type III collagen-positive cells. Establishment of a method for the primary culture of MEEC will provide a new approach for studying the role of epithelial cells in the pathogenesis of various types of otitis media.
本文介绍了一种从龙猫中获取中耳上皮细胞（MEEC）原代培养物的可重复方法。首先用蛋白酶解离MEEC，并将其接种于胶原包被的膜上，使用含有等量杜氏改良 Eagle 培养基和 Ham's F12 且添加 0.5%胎牛血清的培养基进行培养。接种后24小时内首次观察到细胞生长，6 - 7天达到汇合。这些细胞呈单层生长，外观呈卵圆形或多边形。通过免疫荧光显微镜观察，这些细胞细胞角蛋白染色阳性，但III型胶原染色阴性。相反，成纤维细胞III型胶原染色阳性，但细胞角蛋白染色阴性。根据生长特性、形态学和免疫荧光结果，确定这些细胞为上皮细胞。为抑制成纤维细胞生长，在接种后第2天向培养基中添加5 mM腐胺。当将成纤维细胞定义为III型胶原阳性细胞时，其污染率始终低于5%。MEEC原代培养方法的建立将为研究上皮细胞在各种中耳炎发病机制中的作用提供一种新方法。
