Isolation, culture and characterization of primary mouse RPE cells.
Isolation, culture and characterization of primary mouse RPE cells.
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DOI：
10.1038/nprot.2016.065
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发表时间：
2016-07
期刊：
影响因子：
14.8
通讯作者：
Pierce EA
中科院分区：
文献类型：
--
作者：
Fernandez-Godino R;Garland DL;Pierce EA
小鼠模型是研究眼部疾病的有力工具。视网膜色素上皮（RPE）的形态和功能的改变是许多眼部疾病共有的共同特征。我们报告了一个详细的协议，收集，种子，培养和表征视网膜色素上皮细胞从小鼠。我们描述了一种可重复的方法，我们以前开发的收集和培养小鼠RPE细胞transwell作为功能极化单层。RPE细胞的收集需要约3小时，并且培养物在1周内模拟体内RPE细胞特征。该方案还描述了在1-2周内通过透射和扫描电子显微镜（分别为teM和seM）、振动切片机切片和平板的免疫染色以及跨上皮电阻的测量来表征transwell上的细胞的方法。RPE细胞培养物适合于研究来自10天至12个月龄的野生型和遗传修饰的小鼠品系的RPE的生物学。RPE细胞也可以被操作以研究在许多眼部疾病的小鼠模型中RPE病理学的分子机制。此外，体外RPE病理建模代表了一种测试药物的新方法，这将有助于加速黄斑变性（MD）等威胁视力疾病的疗法的开发。
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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DOI：
10.1073/pnas.1130432100
发表时间：
2003-05-27
影响因子：
11.1
作者：
Gibbs, D;Kitamoto, J;Williams, DS
通讯作者：
Williams, DS
影响因子：
7.2
作者：
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通讯作者：
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影响因子：
5.3
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Kay P;Yang YC;Paraoan L
通讯作者：
Paraoan L
影响因子：
4.4
作者：
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通讯作者：
Xu, Heping
影响因子：
3.4
作者：
Johnson, LV;Leitner, WP;Anderson, DH
通讯作者：
Anderson, DH