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

Published: May 24, 2024

doi:

Alyssa Hubal², Amelia Pfaff, Sarah Vos, Mala Upadhyay, Vera Bonilha, Carlos S. Subauste²

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

Summary

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Abstract

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Introduction

The retinal pigment epithelium (RPE) is a specialized cell monolayer lining the Bruch's membrane located between photoreceptors and the choroid¹. RPE cells play a critical role in the proper function of the retina. RPE cells transport glucose and vitamin A to photoreceptors, promote vision by re-isomerization of all-trans retinal into 11-cis retinal and maintain outer segments of photoreceptor through phagocytosis of shed outer segments, remove water from the subretinal space, form the outer blood-retinal barrier through the presence of tight junctions and secrete neurotropic growth factors (such as Pigment Epithelium Derived Factor, and Basic Fibroblast Growth Factor) that support photoreceptors². Dysfunction of RPE cells is involved in the pathogenesis of various retinopathies, including age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy³^,⁴^,⁵. In vitro studies using RPE cells are critical to improving our understanding of the pathogenesis of these diseases. Primary RPE cells are much preferred for these studies since RPE cell lines, while readily available, lack key characteristics of primary RPE cells.

Whereas various species have been utilized as sources of primary RPE cells, mice have the advantage of using genetic modifications to help understand the pathogenesis of retinopathies. Previously described protocols to isolate RPE cells from rodents either require the use of neonatal animals, are lengthy, require technical skill, or are not suitable for culture⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹². We describe a simple and fast method to isolate RPE cells from adult mice that yield highly pure cultures of these cells.

Protocol

The use of animal subjects in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Case Western Reserve University. 1. Reagents preparation Prepare wash buffer medium by supplementing Hank's balanced salt solution (HBSS), no calcium, no magnesium, no phenol red with 10 mM HEPES buffer solution. Keep solution at 4 °C until use. Prepare RPE medium by supplementing Dulbecco's Modified Eagle's Medium with 4.5 …

Representative Results

The described protocol has been used on C57BL/6 background mice. Gender does not appear to change the ability to culture RPE. Mice under 6 weeks yield limited RPE sheets in comparison to older mice, and more eyes may be needed to reach optimal confluency. Following isolation, RPE cells take roughly 3 days to stabilize and attach to the cell culture plate. Approximately 24 h after isolation, round, pigmented cells that appear anucleate have begun to settle but have not fully adhered to the plate (Figu…

Discussion

In this article, we have outlined a simplified protocol for the isolation and culture of murine retinal pigment epithelium. RPE cells isolated from the eyes of adult mice expressed an RPE-specific marker, RPE65, and an intercellular junction marker, ZO-1. Additionally, the cultured cells developed into pigmented, hexagonal sheets in culture.

Several methods for isolation of RPE in rodents have been published previously⁶^,⁷^,<s…

Declarações

The authors have nothing to disclose.

Acknowledgements

Research reported in this publication was supported by NIH Grants R01EY018341 and R01EY019250 (C.S.S.), NIH Grant F31EY035156 (A.H.), and P30 EY011373. The funding organization had no role in the design or conduct of this research.

Materials

0.009 RD Single-Edge Blades	Personna	941202
Dulbecco's Modified Eagle's Medium (DMEM)	Corning	10-013-CV	with 4.5 g/L glucose, L-glutamine, sodium pyruvate
Fetal bovine serum	Corning	35010CV
GlutaMAX, 100x	Gibco	35050061
Hank's Balanced Salt Solution	Gibco	14175095	no Calcium, no magnesium, no phenol red
HEPES Buffer Solution (1M)	Gibco	15630106
MEM Non-Essential Amino Acids, 100x	Gibco	11140050
Micro-Unitome Knife	BVI Beaver	377546
Penicillin-Streptomycin Solution, 100x	Corning	30-002-CI
Polystyrene Microplates	Falcon	08-772-1	24-well or 48-well
Regular Fetal Bovine Serum	Corning	35-010-CV
Trypsin-EDTA (0.25%)	Gibco	25200056	with phenol red
Vannas scissors	Fine Science Tools	10091-12

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