原代小鼠RPE细胞的分离，培养和鉴定。,Nature Protocols

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原代小鼠RPE细胞的分离，培养和鉴定。
Nature Protocols ( IF 13.1 ) Pub Date : 2016-07-01 , DOI: 10.1038/nprot.2016.065
Rosario Fernandez-Godino , Donita L Garland , Eric A Pierce

鼠标模型是研究眼部疾病的有力工具。视网膜色素上皮（RPE）的形态和功能改变是许多眼疾共有的共同特征。我们报告了一个详细的协议，以收集，接种，培养和表征来自小鼠的RPE细胞。我们描述了一种可重现的方法，该方法是我们先前开发的用于在Transwells上收集和培养鼠类RPE细胞作为功能性极化单层膜的方法。RPE细胞的收集需要约3小时，并且培养物可在1周内模拟体内RPE细胞的特征。该协议还描述了通过透射和扫描电子显微镜（分别为TEM和SEM），振动切片和平坦支架的免疫染色以及跨上皮电阻测量来表征1-2周内Transwell细胞的方法。RPE细胞培养物适合研究10 d至12个月大的野生型和转基因小鼠品系的RPE生物学。还可以对RPE细胞进行操作，以研究众多眼部疾病小鼠模型中RPE病理学基础的分子机制。此外，在体外对RPE病理学进行建模代表了一种测试药物的新方法，该方法将有助于加速针对威胁视力的疾病（如黄斑变性（MD））的疗法的开发。

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Isolation, culture and characterization of primary mouse RPE cells.

Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

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