Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

Ryan Tomaszewski; Pragya Rajpurohit; Mei Cheng; Amany Tawfik

doi:10.3791/63543

JoVE Journal
Biology
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JoVE Journal Biology

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

初代マウス網膜色素上皮細胞の単離

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06:21 min

November 4, 2022

DOI: 10.3791/63543-v

Ryan Tomaszewski^1,2, Pragya Rajpurohit³, Mei Cheng^1,2, Amany Tawfik^1,2,3

¹Eye Research Institute,Oakland University, ²Eye Research Center (OUWB)/ERC,William Beaumont School of Medicine, ³Oral Biology and Diagnostic Sciences, Dental College of Georgia,Augusta University

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Overview

This study presents a simplified protocol for isolating retinal pigmented epithelium (RPE) cells from mouse eyes, which are crucial for maintaining photoreceptor function within the retina. The stepwise method includes eye enucleation, dissection, and the subsequent isolation and culturing of RPE cells, aimed at facilitating research on retinal conditions.

Key Study Components

Research Area

Cell biology
Retinal health and diseases
Isolation techniques

Background

RPE cells support photoreceptor maintenance and function.
Primary RPE isolation is a critical tool for studying the outer blood-retinal barrier.
Relevance to conditions such as age-related macular degeneration.

Methods Used

Dissection and enzymatic treatment of mouse eyes.
Mouse model system.
Cell culture and validation techniques (RPE65 marker).

Main Results

Successful isolation of RPE cells with maintained pigmentation and viability.
Demonstrated integrity of RPE cells validated by specific markers.
Transmission of a simplified adaptation of existing isolation protocols.

Conclusions

This study outlines an effective method for RPE cell isolation.
The protocol can enhance research regarding retinal diseases and cellular functions in biology.

Frequently Asked Questions

What is the significance of RPE cells?

RPE cells are essential for the health of photoreceptor cells and overall retinal function.

How are RPE cells typically isolated?

They are isolated through a combination of dissection techniques and enzymatic treatment of mouse eyes.

What diseases are associated with RPE dysfunction?

Age-related macular degeneration is a significant condition related to RPE dysfunction.

Can this protocol be used in various research settings?

Yes, the protocol is designed to utilize common materials available in most labs.

How are RPE cells validated after isolation?

Validation is performed using specific markers such as RPE65 and cytoskeletal proteins.

What is the optimal storage condition for isolated RPE cells?

Isolated RPE cells can be incubated in complete growth media at 37 degrees Celsius.

What are the benefits of this simplified protocol?

It provides a more accessible method for researchers studying RPE cells and related retinal diseases.

この原稿では、網膜色素上皮(RPE)細胞をマウスの目から段階的に単離するための簡略化されたプロトコルについて説明しています。このプロトコルには、マウスの目の除核と解剖、それに続くRPE細胞の単離、播種、培養が含まれます。

このプロトコルでは、初代マウス網膜色素性上皮細胞を単離するための簡単で効率的な方法を概説します。網膜色素上皮細胞は、視細胞を維持し、網膜の正常な機能を確保するために非常に重要な役割を果たしています。当研究室では、初代マウス色素上皮細胞を、網膜外関門や加齢黄斑変性症などの関連疾患を研究するための重要なツールとして分離しています。

マウスの目を抽出するには、施設の承認された方法に従ってマウスを倫理的に安楽死させ、マウスを滅菌アンダーパッドに置き、眼窩領域の5/45スタイルのピンセットを押して眼球を核形成してプロプトーシスを誘発し、ピンセットを目の後部に移動し、視神経がまだ無傷であることを確認するためにゆっくりと引っ張って目を取り除きます。鉗子を使用しながら、5%ポビドンヨウ素を含む2ミリリットルの微量遠心チューブに目を浸します。室温で2〜3分間目をインキュベートします。

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