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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

Validation of the specificity, purity, and barrier function of isolated Müller cells
To confirm the viability, morphology, and distinctive qualities of the isolated Müller cells, the cells were examined under a light microscope. P0 and P1 images were recorded (Figure 1A). To check the contamination of isolated Müller cells with RPE cells and confirm it's purity, immunofluorescence staining (IF) was performed using antibodies specific to RPE cell.......

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

Acknowledgements

This work was supported by National Eye Institute (NEI),The National Eye Institute (NEI) fund R01 EY029751-04. We would like to acknowledge Dr. Sylvia B. Smith as this protocol was modified version based on her protocol of Müller cell isolation.

....

Materials

NameCompanyCatalog NumberComments
Beaker : 100mLKIMAX14000
Collagenase IV Worthington LS004188
Disposable Graduated Transfer Pipettes :3.2mL Sterile13-711-20
DMEM (1X) Thermo Scientific11885084Media to grow Müller cells 
Fetal Bovine Serum (FBS)gibco26140079For complete Muller cell culture media
Glutamine synthaseCell signalling80636
Heracell VISO 160i CO2 IncubatorThermo Scientific50144906
KimwipesKimberly-Clark34155
Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mLB-D309577
Micro Centrifuge Tube: 2 mLGrainger11L819
Pen Strepgibco15140-122For complete Müller cell culture media
Phosphate Buffer saline (PBS)Thermo ScientificJ62851.AP
Positive Action Tweezers, Style 5/45Dumont72703-DZ
Scissors Iris Standard Straight 11.5cmGARANA INDUSTRIES2595
Sorvall St8 CentrifugeThermoScientific75007200
Stemi 305 MicroscopeZeissn/a
Surgical Blade, #11, Stainless SteelBard-Parker371211
Suspension Culture Dish 60mm x 15mm StyleCorning430589
Tissue Culture Dish : 100x20mm styleCorning353003
Tornado Tubes: 15mLMidsciC15B
Tornado Tubes: 50mLMidsciC50R
Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm TipsDumont501764
Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OALElectron Microscopy Sciences Dumont50-241-57
Underpads, Moderate : 23" X 36"McKesson4033
Vannas Spring Scissors - 2.5mm Cutting EdgeFST15000-08
Vimentin invitrogenMA5-11883
Zeiss AxioImager Z2Zeissn/a
Zeiss Zen Blue 2.6Zeissn/a

References

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