A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Alyssa Hubal; Amelia Pfaff; Sarah Vos; Mala Upadhyay; Vera Bonilha; Carlos S. Subauste

doi:10.3791/66921

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

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05:04 min

May 24, 2024

DOI: 10.3791/66921-v

Alyssa Hubal^1,2, Amelia Pfaff², Sarah Vos², Mala Upadhyay³, Vera Bonilha³, Carlos S. Subauste^1,2

¹Department of Pathology,Case Western Reserve University, ²Division of Infectious Diseases and HIV Medicine, Department of Medicine,Case Western Reserve University, ³Cole Eye Institute, Ophthalmic Research,Cleveland Clinic

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Overview

The study presents a simple and effective protocol for isolating and culturing adult murine retinal pigment epithelium (RPE). RPE serves as a crucial barrier between the choroid and retina, playing a key role in the health of retinal cells, especially photoreceptors.

Key Study Components

Area of Science

Neuroscience
Cell biology
Retinal health

Background

RPE is essential for maintaining retinal cell function.
Dysfunction of RPE is involved in diseases like age-related macular degeneration.
Mice provide a model for genetic manipulation to study retinopathies.
Current isolation methods for RPE are often lengthy and require technical expertise.

Purpose of Study

To develop a fast and straightforward protocol for isolating primary RPE from adult mice.
To enable high yield and purity of cultured RPE for research applications.
To provide a foundation for studying disease development in the retina.

Methods Used

The main method involves isolating RPE from adult mice through dissection.
A surgical approach is used to remove the eye, followed by enzymatic digestion.
RPE is cultured in a specific medium for observation and analysis post-isolation.
Critical steps include eye dissection, trypsin incubation, and centrifugation.
Cell appearance and adherence are monitored over several days post-culture.

Main Results

The protocol yields highly pure RPE cultures suitable for long-term studies.
RPE cells show distinct morphological changes and increase in confluency over time.
Cells exhibit characteristic pigmentation and form polarized monolayers.
Results confirm integrity of cultured RPE, supporting their use in further research.

Conclusions

The study provides an accessible method to isolate adult murine RPE, facilitating research into retinal diseases.
The protocol enhances the understanding of cellular mechanisms related to RPE health and disease.
Potential applications include investigations into age-related macular degeneration and other retinal pathologies.

Frequently Asked Questions

What are the advantages of this isolation protocol?

This protocol is straightforward and does not require specialized skills or extensive time, making it accessible for researchers.

How is the primary RPE isolated from adult mice?

RPE is isolated through surgical dissection of the eye, followed by enzymatic digestion to release the RPE sheets.

What types of data can be obtained from cultured RPE cells?

Researchers can observe morphological characteristics, molecular markers, and cell viability over time to assess RPE health.

Can this method be adapted for other species?

While this method is optimized for mice, it may be adaptable for other species with appropriate protocol modifications.

What are the limitations of this isolation method?

The method may not be suitable for obtaining RPE from older or diseased animals, as cell quality could be compromised.

How does the RPE culture change over time?

Over 48-72 hours, RPE cells exhibit changes in adherence and confluency, leading to the formation of a polarized monolayer.

網膜色素上皮(RPE)は、脈絡膜と網膜との間の重要な障壁として機能し、光受容体などの網膜細胞タイプの健康と機能を促進します。ここでは、成体マウスRPEを単離および培養するための簡単で効果的なプロトコルについて説明します。

網膜色素上皮細胞またはRPEは、光受容体と脈絡膜の間に位置しています。RPEの機能不全は、加齢性黄斑変性症、糖尿病性網膜症、網膜色素変性症などの疾患の病因において重要です。一次RPEの使用は、疾患がどのように発症するかについての理解を深めるための研究の鍵です。

一次RPEの発生源としてさまざまな種が使用されていますが、マウスには、網膜症がどのように発症するかを理解するために遺伝子操作を使用できるという利点があります。げっ歯類から一次RPEを単離する従来の方法は、新生児動物の使用が必要であったり、長期にわたるものであったり、技術的な専門知識が必要であったり、細胞培養に適さなかったりします。成体マウスから一次RPEを単離し、細胞の高純度培養物を得るための簡単で迅速な方法について説明します。

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