Isolation of Primary Mouse Retinal Glial Müller Cells

Summary

This manuscript describes a detailed protocol for isolating retinal glial Müller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of Müller cells.

Abstract

The primary supporting cell of the retina is the retinal glial Müller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, Müller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse Müller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, Müller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for Müller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn't be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

Introduction

Müller cells (MCs) are the main and most abundant glial cells found in the retinal tissue. They are the key players in providing structural integrity and metabolic functions within the retina¹. The strategic structure of MCs is spread across the entire retina thickness, thus providing support to the retina. In addition to their scaffold-like properties, they have metabolic functions for retinal neurons, supplying them with energy substrates, including glucose and lactate. These functions are crucial in order to maintain healthy neuronal function. Impaired MCs have been reported to contribute to various retinal diseases, including age-relat....

Protocol

All experiments with animals conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were done following our animal protocol approved by the Institute for Animal Care and Use Committee (IACUC) and Oakland University policies (protocol number 2022-1160)

1. Media and solution preparation

1. Prepare Müller cell eye solution by supplementing the Dulbecco Modified Eagle Medium (DMEM, details in the material table) with penicillin/s.......

Discussion

The isolation of primary retinal pigmented epithelium (RPE) from mice was previously documented by our lab. This manuscript describes a detailed demonstration protocol for primary Müller cells isolation. This procedure involves enucleation, treatment, dissection, collection, seeding, culture, and characterization of Müller cells isolated from mouse eyes. It is based on a previously successful protocol found in earlier publications and our modified protocol that we used in a recent publication

Materials

Name	Company	Catalog Number	Comments
Beaker : 100mL	KIMAX	14000
Collagenase IV	Worthington	LS004188
Disposable Graduated Transfer Pipettes :3.2mL Sterile		13-711-20
DMEM (1X)	Thermo Scientific	11885084	Media to grow Müller cells
Fetal Bovine Serum (FBS)	gibco	26140079	For complete Muller cell culture media
Glutamine synthase	Cell signalling	80636
Heracell VISO 160i CO2 Incubator	Thermo Scientific	50144906
Kimwipes	Kimberly-Clark	34155
Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL	B-D	309577
Micro Centrifuge Tube: 2 mL	Grainger	11L819
Pen Strep	gibco	15140-122	For complete Müller cell culture media
Phosphate Buffer saline (PBS)	Thermo Scientific	J62851.AP
Positive Action Tweezers, Style 5/45	Dumont	72703-DZ
Scissors Iris Standard Straight 11.5cm	GARANA INDUSTRIES	2595
Sorvall St8 Centrifuge	ThermoScientific	75007200
Stemi 305 Microscope	Zeiss	n/a
Surgical Blade, #11, Stainless Steel	Bard-Parker	371211
Suspension Culture Dish 60mm x 15mm Style	Corning	430589
Tissue Culture Dish : 100x20mm style	Corning	353003
Tornado Tubes: 15mL	Midsci	C15B
Tornado Tubes: 50mL	Midsci	C50R
Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips	Dumont	501764
Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL	Electron Microscopy Sciences Dumont	50-241-57
Underpads, Moderate : 23" X 36"	McKesson	4033
Vannas Spring Scissors - 2.5mm Cutting Edge	FST	15000-08
Vimentin	invitrogen	MA5-11883
Zeiss AxioImager Z2	Zeiss	n/a
Zeiss Zen Blue 2.6	Zeiss	n/a