Freie Universität Berlin, Institute of Veterinary Biochemistry, Oertzenweg 19b, Berlin, Germany.
BMC Vet Res. 2012 Mar 19;8:31. doi: 10.1186/1746-6148-8-31.
Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue.
We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining.
We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.
宫颈阴道上皮细胞在雌性哺乳动物生殖道中维持生理和无病原体环境，并参与精子-上皮相互作用。目前还没有易于获得的、分化的宫颈上皮模型系统来阐明这些高度特化细胞中的潜在分子机制。因此，本研究旨在建立一种猪宫颈上皮细胞培养体系，以模拟组织的体内特性。
我们测试了不同的分离方法和培养条件，并通过免疫组织化学方法对角蛋白进行了培养细胞纯度的验证。我们能够从宫颈组织外植体中重复性地培养出纯上皮细胞。基于形态评分和 WST-1 增殖测定，我们优化了生长培养基的组成。原代猪宫颈细胞在含有 10% FCS、EGF 和胰岛素的条件化 Ham's F-12 中表现最佳。在气液界面培养三周后，细胞呈现出不连续的多层表型。最后，通过免疫组织化学方法对β-连环蛋白进行了分化验证。通过阿尔辛蓝染色可以显示粘多糖的产生。
我们提供了第一个基于屠宰场材料的、使用易于获得的细胞建立分化的猪宫颈上皮模型的合适方案。
