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

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 retinopathy^3,4,5. 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 culture^{6,7,8,9,10,11,12}. 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

1. 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.
2. Prepare RPE medium by supplementing Dulbecco's Modified Eagle's Medium with 4.5 g/L glucose, 1.25x GlutaMAX Supplement (stock concentration 100x or 200 mM; final concentration of 2.5 mM L-glutamine), with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1x MEM Non-Essential Amino Acids Solution (100x). Before use, pre-warm the media to 37 °C in a water bath.

2. Mouse eye extraction

1. Euthanize the mouse, preferably ranging from 3- to 14-weeks in age, using an IACUC-approved method of euthanasia (cervical dislocation under anesthesia using a Ketamine/xylazine cocktail at 0.1 mL per 20 g of mouse [87.5 mg/kg Ketamine and 12.5 mg/kg Xylazine] was the method used in this study).
   NOTE: Eyes collected from younger mice will facilitate increased retinal pigment epithelial cell yield over time and extend the capacity for successive passages.
2. Lay the mouse flat on its side with the eye oriented upwards.
3. Place the index finger and thumb above and below the eye, respectively. Gently apply pressure on the bone structure surrounding the eye to induce protrusion of the eyeball.
4. Insert the tip of slightly opened scissors underneath the eye and gently rotate the wrist away from the eye 90° until the eye detaches from the socket.
   NOTE: Do not close the scissors to cut the eye or the optic nerve, as it will make dissection of the eyecup more difficult.
5. Immediately place and roll the eye in 70% ethanol for no more than 5 s before transferring it to a wash buffer medium kept on ice.
6. Under a dissecting microscope, transfer one eye at a time to a Petri dish filled with wash buffer and strips of soaked gauze.
7. Stabilize the eye by holding the optic nerve with tweezers. Gently make an incision at the level of the ora serrata using a 3.00 mm 45° surgical knife or 0.009" razor blade.
8. Using Vannas scissors, gently insert the scissor into the incision and cut around the circumference of the ora serrata until the anterior segment and vitreous can be removed and discarded.
9. Gently peel away the retina from the eyecup using tethered forceps, careful not to disturb the RPE layer.
   NOTE: To make the retina easier to remove, fill a transfer pipette with wash buffer medium and carefully apply it to the edges of the eyecup to gently lift the retina.
10. Finally, remove the optic nerve and excess connective tissue from the eyecup using scissors. Be careful not to puncture the eyecup.

3. Isolation of primary RPE

1. Transfer the eyecup to a 1.5 mL microcentrifuge tube containing 1 mL of 0.25% trypsin + 0.02% EDTA.
   NOTE: Two eyecups can be added to one aliquot of Trypsin-EDTA to increase the yield of culturable RPE.
2. Transfer microcentrifuge tubes to a water bath set at 37 °C and incubate for 10 min. Every 2 min, remove the tubes from the water bath and firmly tap the bottom of the tube 40 times onto the countertop.
3. After 10 min, gently disrupt the eyecup mechanically using a P1000 or a 2 mL serological pipet by pipetting up and down no more than 3 times.
   NOTE: Fragmentation of the RPE sheets is essential as large sheets will not adhere properly.
4. Neutralize the trypsin immediately by layering the detached RPE sheets, but not the eyecup, onto 0.5 mL of fetal bovine serum (FBS) in a 15 mL conical tube.
5. Further, dilute the trypsin by layering 3 mL of RPE media dropwise onto the layers of FBS and RPE sheets.
6. Centrifuge the RPE at 340 x g for 3 min.
7. Discard the supernatant and resuspend cells in a suitable amount of RPE media for either a 24-well plate (1.9 cm²/well) or a 48-well plate (0.75 cm²/well).
   NOTE: RPE can be plated onto well plates coated with extracellular matrix proteins, specifically laminin, collagen IV, or fibronectin, to increase adherence¹¹. RPE can also be resuspended in 1 mL of RPE media and layered onto a 40% density separation gradient to further isolate pure RPE cells. Additionally, spinning the plate at 340 x g for 3-5 min may increase cell adhesion¹³.
8. Incubate at 37 °C at 5% CO₂.

4. Culturing RPE

1. Do not disrupt the isolated RPE for at least 3 days. After 72 h, gently remove the old medium and replace it with fresh, pre-warmed media.
2. Change the medium every 48 h after the first 3 days.
3. Once cells reach confluency, passage the cells using 0.25% trypsin + 0.02% EDTA and reduce the FBS in the RPE media to 2%.
   NOTE: Primary RPE were previously reported to begin de-differentiation after 5-7 passages and will begin to lose their hexagonal shape and pigmentation¹⁴.

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 previously^6,7,

Materials

Name	Company	Catalog Number	Comments
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