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).
Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Rosario
R
Fernandez-Godino
Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Donita L
DL
Garland
Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Eric A
EA
Pierce
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D017097:Electric Impedance; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000648:ultrastructure