Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Blanca Chinchilla; Heran Getachew; Rosario Fernandez-Godino

doi:10.3791/62228

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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Biology

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JoVE Journal Biology

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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08:33 min

February 10, 2021

DOI: 10.3791/62228-v

Blanca Chinchilla¹, Heran Getachew¹, Rosario Fernandez-Godino¹

¹Ocular Genomics Institute of Massachusetts Eye and Ear,Harvard Medical School

Overview

This protocol outlines the efficient isolation and culture of mouse retinal pigment epithelium (RPE) cells, which are critical for studying eye diseases such as age-related macular degeneration. Within one week, a functional and polarized RPE monolayer can be established, providing valuable insights into underlying disease mechanisms.

Key Study Components

Research Area

Cell biology
Ophthalmology
Pathobiology of eye diseases

Background

Mouse RPE cultures serve as models for understanding eye disease mechanisms.
Age-related macular degeneration is a leading cause of blindness in the elderly.
Proper dissection techniques are crucial for successful RPE cell isolation.

Methods Used

Isolation and culture of mouse RPE cells using a stepwise dissection and incubation protocol.
Mouse as the biological system.
Use of trypsin-EDTA and FBS solutions in cell culture preparation.

Main Results

A polarized RPE monolayer was achieved, confirmed by morphology and functional assays.
High transepithelial resistance (TER) values were obtained, indicating successful polarization.
Phagocytosis assays demonstrated functional characteristics of the RPE cells.

Conclusions

This study provides a reliable protocol for isolating mouse RPE cells as a model for eye disease research.
The findings contribute to the understanding of RPE cell biology and its implications for ophthalmic disease research.

Frequently Asked Questions

What are the applications of mouse RPE cell cultures?

They can be used to study the mechanisms of various eye diseases, including age-related macular degeneration.

How long does the culture process take?

The protocol allows for the establishment of a functional RPE monolayer within one week.

What is critical for successful RPE cell isolation?

Proper dissection techniques and careful handling of the eyeballs are essential.

How is the polarization of RPE cells verified?

Polarization is verified through transepithelial resistance (TER) measurements.

Can this protocol be used in other research models?

While this protocol is specific to mouse RPE cells, similar techniques may be adapted for other organisms.

What reagents are needed for the protocol?

Key reagents include HEPES-free HBSS buffer, trypsin-EDTA, and FBS solutions.

How do RPE cells respond in functional assays?

They exhibit normal morphological features and demonstrate phagocytic activity.

This protocol, which was originally reported by Fernandez-Godino et al. in 2016¹, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

This protocol allows for the efficient isolation and culture of mouse RPE cells. Healthy primary mouse RPE cultures are valuable models to understand the mechanisms of underlying eye diseases. Confluent and viable RPE cultures obtain within three days and can be grown for weeks.

This protocol has been successfully used to understand the mechanisms underlying early age-related macular degeneration, which is the leading cause of blindness among the elderly in developed countries. It is important to visualize the dissection steps. Details are critical for the success of the protocol, for instance, how to handle the eyeballs so that the sclera isn't punched, where exactly cuts should be performed, and how to embed the eye cups in trypsin so that they stay open.

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