Isolation, culture and characterization of primary mouse RPE cells

• Published in: Nature protocols Vol. 11; no. 7; pp. 1206 - 1218
• Main Authors: Fernandez-Godino, Rosario, Garland, Donita L, Pierce, Eric A
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-07-2016; Nature Publishing Group
• Subjects: 14/19; 14/28; 631/1647/1407/651; 631/1647/767/1424; 631/80/85/2361; 692/699/3161; Analytical Chemistry; Animals; Biological Techniques; Care and treatment; Cell Culture Techniques - methods; Cell Separation - methods; Cells, Cultured; Computational Biology/Bioinformatics; Drugs; Electric Impedance; Genetic engineering; Immune system; Immunohistochemistry - methods; Life Sciences; Macular degeneration; Methods; Mice; Microarrays; Microscopy, Electron, Scanning - methods; Microscopy, Electron, Transmission - methods; Organic Chemistry; Pathology; protocol; Retina; Retinal pigment epithelium; Retinal Pigment Epithelium - cytology; Retinal Pigment Epithelium - ultrastructure; Scanning microscopy; United States > US; Massachusetts
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.065

This protocol describes the isolation and culture of primary mouse RPE cells from various mouse models to produce viable RPE cells
in vitro
that mimic
in vivo
characteristics
This protocol describes the isolation and culture of primary mouse RPE cells from various mouse models to produce viable RPE cells
in vitro
that mimic
in vivo
characteristics
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).