Human Retinal Pigment Epithelial Cell Cultures: Physiology & Fluid Transport

眼部疾病如年龄相关性黄斑变性或糖尿病影响视网膜色素上皮（RPE）功能并导致视网膜变性、视力丧失和失明。为了研究RPE的功能、生理学和病理学，许多实验室试图培养RPE作为天然组织的更易获得的替代物。由于RPE及其在视网膜和脉络膜中的相邻细胞的功能和形态复杂性，该目标已经以不同程度的成功实现。 我们已经开发了用于培养人胎儿RPE细胞的汇合单层的技术，所述细胞表现出与天然人胎儿RPE相似的形态、生理学和蛋白质表达模式。 这项工作的目标之一是确定一组市售成分，以创建稳定和可重复的RPE细胞培养物。我们能够产生融合的色素RPE细胞培养物，其具有经典的上皮形态、1 - 3 mV的跨上皮电位和大于400 Ohms* cm 2的跨上皮电阻。 在本实验中，我们进一步的特点，这些文化，使用电子显微镜和免疫组化，以确定细胞结构，并定位顶端和基底外侧膜和细胞间连接复合物蛋白。 ELISA用于确认所选细胞因子的分泌极性。 细胞内微电极被用来表征受体介导的第二信使途径及其下游的电生理特性，在顶端和基底外侧膜。 电容探针技术用于测量净跨上皮液体转运。 该模型已被用于帮助确定一些介导氧化应激和硫辛酸保护作用的线粒体途径。定义了RPE的基因签名

Eye diseases such as age-related macular degeneration or diabetes affect retinal pigment epithelium (RPE) function and lead to retinal degeneration, vision loss and blindness. To study RPE function, physiology, and pathology, many laboratories have attempted to culture RPE as a more accessible alternative to native tissue. This goal has been accomplished with varying degrees of success due to the functional and morphological complexity of the RPE and its neighboring cells in the retina and the choroid. We have developed techniques for culturing confluent monolayers of human fetal RPE cells that exhibit morphology, physiology, and patterns of protein expression similar to native human fetal RPE. One of the goals of this work was to identify a set of commercially available ingredients to create stable and reproducible RPE cell cultures. We been able to produce confluent pigmented RPE cell cultures with classic epithelial morphology, transepithelial potential of 1 - 3mV, and transepithelial resistance greater than 400 Ohms*cm2. In the present experiments we further characterized these cultures using electron-microscopy and immunohistochemistry to identify cell structures, and localize apical and basolateral membrane and intercellular junctional complex proteins. ELISAs were used to confirm the polarity of secretion of selected cytokines. Intracellular microelectrodes were used to characterize receptor-mediated second messenger pathways and their downstream electrophysiological properties at the apical and basolateral membranes. The capacitance probe technique was used to measure net transepithelial fluid transport. This model has been used to help identify some of the mitochondrial pathways that mediate oxidative stress and the protective effects of lipoic acid. Gene signature of RPE was defined