Blenkinsop Timothy A, Salero Enrique, Stern Jeffrey H, Temple Sally
Neural Stem Cell Institute, Rensselaer, NY, USA.
Methods Mol Biol. 2013;945:45-65. doi: 10.1007/978-1-62703-125-7_4.
The retinal pigment epithelium (RPE) is implicated in many eye diseases, including age-related macular degeneration, and therefore isolating and culturing these cells from recently deceased adult human donors is the ideal source for disease studies. Adult RPE could also be used as a cell source for transplantation therapy for RPE degenerative disease, likely requiring first in vitro expansion of the cells obtained from a patient. Previous protocols have successfully extracted RPE from adult donors; however improvements in yield, cell survival, and functionality are needed. We describe here a protocol optimized for adult human tissue that yields expanded cultures of RPE with morphological, phenotypic, and functional characteristics similar to freshly isolated RPE. These cells can be expanded and cultured for several months without senescence, gross cell death, or undergoing morphological changes. The protocol takes around a month to obtain functional RPE monolayers with accurate morphological characteristics and normal protein expression, as shown through immunohistochemistry analysis, RNA expression profiles via quantitative PCR (qPCR), and transepithelial resistance (TER) measurements. Included in this chapter are steps used to extract RPE from human adult globes, cell culture, cell splitting, cell bleaching, immunohistochemistry, and qPCR for RPE markers, and TER measurements as functional test.
视网膜色素上皮（RPE）与许多眼部疾病有关，包括年龄相关性黄斑变性，因此从近期死亡的成年人类供体中分离和培养这些细胞是疾病研究的理想来源。成年RPE也可作为RPE退行性疾病移植治疗的细胞来源，这可能首先需要对从患者获得的细胞进行体外扩增。先前的方案已成功从成年供体中提取RPE；然而，仍需要提高产量、细胞存活率和功能。我们在此描述一种针对成人组织优化的方案，该方案可产生具有与新鲜分离的RPE相似的形态、表型和功能特征的RPE扩增培养物。这些细胞可以扩增和培养数月而不会衰老、大量细胞死亡或发生形态变化。该方案大约需要一个月的时间来获得具有准确形态特征和正常蛋白质表达的功能性RPE单层，这通过免疫组织化学分析、定量PCR（qPCR）的RNA表达谱以及跨上皮电阻（TER）测量得以证明。本章包括从成人眼球中提取RPE、细胞培养、细胞传代、细胞漂白、免疫组织化学、RPE标志物的qPCR以及TER测量作为功能测试所使用的步骤。
