Related Experiment Video
Updated: Mar 19, 2026
Isolation of Primary Mouse Retinal Pigmented Epithelium Cells
Published on: November 4, 2022
Isolation, culture and characterization of primary mouse RPE cells
Rosario Fernandez-Godino1, Donita L Garland1, Eric A Pierce1
1Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Abstract:
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).
Insights
This study presents a reproducible protocol for culturing mouse retinal pigment epithelium (RPE) cells. These functional RPE cell cultures aid in studying ocular diseases and developing new therapies for vision disorders.
Area of Science:
- Ophthalmology
- Cell Biology
- Translational Medicine
Background:
- Retinal pigment epithelium (RPE) dysfunction is central to many ocular diseases.
- Mouse models are crucial for studying these conditions.
- Standardized RPE cell culture protocols are needed for robust research.
Purpose of the Study:
- To detail a reproducible protocol for collecting, culturing, and characterizing mouse RPE cells.
- To establish functional, polarized RPE monolayers in vitro.
- To provide a platform for studying RPE biology and pathology in ocular disease models.
Main Methods:
- Collection and culture of murine RPE cells on Transwells.
- Characterization using transmission and scanning electron microscopy (TEM/SEM).
- Immunostaining of RPE sections and flat mounts.
- Measurement of transepithelial electrical resistance (TEER).
Main Results:
- Established a protocol yielding functional, polarized RPE monolayers within 1 week.
- Demonstrated successful characterization of cultured RPE cells using multiple methods.
- Confirmed suitability of cultures for studying RPE from various mouse strains and ages.
Conclusions:
- This protocol provides a reliable method for generating functional mouse RPE cell cultures.
- These cultures serve as a valuable tool for investigating RPE pathology in ocular diseases.
- In vitro RPE modeling facilitates drug testing and accelerates therapeutic development for vision-threatening conditions like macular degeneration.
