Institute of Veterinary Physiology, Faculty of Veterinary Medicine, Leipzig University, Leipzig, Germany.
Cells Tissues Organs. 2011;194(6):457-68. doi: 10.1159/000323916. Epub 2011 Mar 9.
Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness.
Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER.
The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1.
The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.
上皮细胞原代培养是研究上皮功能的合适模型，尤其是体外研究上皮紧密性的调节。我们的研究目的是开发一种分离和培养猪结肠上皮细胞的方案，并建立跨上皮电阻（TEER）作为上皮紧密性的功能参数。
通过酶消化Dispase 从仔猪近端结肠中获得上皮细胞。将细胞培养在胶原涂层膜载体上 21 天。通过细胞角蛋白和封闭蛋白 1（ZO-1）的免疫组织化学检测来证明细胞的上皮来源。扫描电子显微镜、透射电子显微镜和共聚焦显微镜用于进一步的形态学特征描述。通过测量 TEER 来确定人工上皮的完整性和紧密性。
培养的上皮细胞对细胞角蛋白和 ZO-1 呈免疫反应性。它们的顶膜上有密集的微绒毛，底膜上有 Na(+)/K(+)-ATP 酶的表达。相邻细胞通过紧密连接连接。我们观察到 TEER 在培养期间持续增加，达到 870±38 Ω·cm(2)。TEER 与表达 ZO-1 的上皮细胞数量相关。
原代培养的上皮细胞的特性类似于体内极化结肠上皮的结构特性。TEER 的测量似乎适用于体外研究上皮紧密性。我们建议使用这些原代上皮培养物来研究上皮屏障功能的调节。
