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

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.

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 (Figure 1A). Over the next 48-72 h, the cells begin to attach and spread while maintaining their dark pigmentation and transparent nucleus (Figure 1B,C). After 5 days, RPE have increased confluency and have started to form cell-to-cell contacts reminiscent of a polarized monolayer with a hexagonal shape (Figure 1D).

Isolated RPE were assessed for purity and tight-junction integrity after 6 days in culture. RPE were first assessed for the presence of retinoid isomerohydrolase or retinal pigment epithelium 65 kDA protein (RPE65), a marker specific for RPE (Figure 2, green). In addition, for RPE to form a single layer of pigmented and hexagonal-shaped cells, the cell-to-cell junctions must remain intact. Tight junction protein-1 or zonula occludens-1 (ZO-1) are scaffolding proteins vital for the maintenance and integrity of the tight junctions between individual RPE cells¹⁴. Confluent isolated RPE cultures maintained their intercellular junction proteins, demonstrated by the staining of ZO-1 found on the periphery of the cell (Figure 2, red). Once these cells reach confluency in large sheets, they maintain cell-to-cell contacts without overgrowing for up to 2 weeks. Previous studies have shown that ZO-1 is expressed highly for up to 9 days in culture¹⁵. Additional markers used in cultures of primary RPE include Collagen IV, CRALBP, and OTX2¹¹.

Figure 1. Morphology of mouse primary RPE cultured on standard polystyrene 24-well plate. (A) Primary RPE isolated from both eyes of a C57BL/6 mouse and cultured in a single well of a non-coated tissue culture polystyrene 24-well plate directly after isolation. (B,C) After 48 h and 72 h, primary RPE begin to adhere to the plate and form pigmented, binucleated cells. (D) After 5 days, brightfield microscopy shows the hyperpigmented, hexagonal-shaped cells increasing confluency and beginning to form cell-to-cell contacts. Scale bar: 100 µm. Please click here to view a larger version of this figure.

Figure 2. RPE-specific marker, RPE-65, and intercellular junction marker, ZO-1, are conserved in isolated primary murine RPE. Primary RPE from three C57BL/6 mice were cultured for 6 days prior to fixation with 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton-X in PBS for 5 min, blocked with 5% normal goat serum, and incubated with antibodies against RPE-65 (green) or ZO-1 (red). Scale bar: 50 µm. Please click here to view a larger version of this figure.

Figure 3. Simplified schematic representation of the retinal pigment epithelium isolation protocol. After removing the eyeball from the mouse, sterilize the eye by dipping it briefly in 70% ethanol before moving to the wash buffer. In the wash buffer, cut along the ora serrata and remove the anterior segment. Remove the retina and place the eyecup in a tube with Trypsin/EDTA. Gently tap the tube on the counter every 2 min for 10 min total, transfer the supernatant to a 15 mL conical tube, and centrifuge the pellet of RPE. Resuspend the cells and plate onto a 24- or 48- well plate. Graphics generated using BioRender. Please click here to view a larger version of this figure.

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^{6,7,8,9,10,11,12}. Many protocols peel the RPE layers from the Bruch's membrane requiring technical skill and increasing the risk of RPE damage or cell death^{6,7,9,11,12,16}. Thus, this protocol utilizes enzymatic detachment of the RPE layer, rather than mechanical dissociation, to promote survival and maintain the integrity of the RPE sheets in culture. This method yields mouse RPE in a culture that maintain RPE morphology with highly pigmented and hexagonal-shaped cells that conserve the expression of tight junction proteins. In addition, this protocol lacks the use of complex reagents or materials used in other protocols, such as permeable membrane inserts, additional extracellular matrix coating proteins, or specialized tissue dissociation reagents. Instead, this protocol utilizes simple enzymatic digestion using 0.25% Trypsin-EDTA in combination with tissue agitation to dissociate the RPE layer from Bruch's membrane. This protocol has been successfully performed on mice aged 3- to 14-weeks-old; however, more eyes may be needed to successfully gain a confluent culture in young mice.

Several challenges may arise during the execution of this protocol. Technical skill is required to precisely cut around the ora serrata and carefully remove the retina without disrupting the RPE layer. The retina may be disturbed while cutting the circumference of the sclera, which can alter RPE viability. Refinement of this technique can only be accomplished through repetitive, continuous practice. Additionally, RPE sheets may remain attached to the retinal layer after removal. To avoid excess loss of RPE, stimulate retinal detachment by using a syringe or transfer pipette filled with wash buffer medium to gently lift the edges of the retina from the eyecup. Hyaluronidase incubation can also facilitate detachment¹⁶. In the event that RPE are not properly detaching from the Bruch's membrane and choroid, increase incubation time with trypsin in the water bath. Additionally, freeze aliquots of fresh trypsin at -20 °C and thaw directly before use to maintain optimal enzymatic activity. Failure of the RPE to attach to the well plate after isolation can arise if cells are seeded at too low of a density or if the RPE were damaged during the isolation process. In addition, our protocol does not require the coating of extracellular matrix (ECM) proteins for culture. However, ECM proteins, such as fibronectin, collagen IV, laminin, or collagen I, have been shown to increase cell attachment and should be considered if adherence is poor¹¹.

To increase the yield of RPE-cultured monolayers, pool at least 2-3 eyes of age-matched mice with the same genetic background; cells can lose their hexagonal shape and pigment over time if not seeded at a high enough density. For mice that are over 8 weeks of age, 2 eyes per 24-well plate should be sufficient for optimal confluency. If mice are under the age of 8 weeks, 3-4 eyes will result in better adherence and confluency. Over time, cells may be susceptible to epithelial-mesenchymal transition (EMT) with loss of pigmentation and acquisition of an elongated shape. The addition of Rho-Kinase and TGFβR-1/ALK5 inhibitors, such as Y27632 and Repsox, respectively, can prevent EMT of the cultured RPE cells¹². While the isolation protocol is brief, only requiring 1 h for isolation of 2-4 eyes, the primary culture may take up to 7-10 days to reach full confluency. In addition, passaging RPE is limited, with an increased risk of EMT at every subsequent passage.

Retinal pigment epithelial cells are critical cells for maintaining homeostasis in the eye. RPE act as phagocytes to aid in the maintenance of photoreceptors, prevent neural layers from light damage, and act as a tight epithelial barrier to regulate transport¹⁷. Diseases directly affecting RPE, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy, can exacerbate inflammation, increase apoptosis, and disrupt cellular junctions, leading to the increased probability of retinal degeneration and blindness¹⁷. Cultivation of primary RPE facilitates the study of the pathogenesis of ocular diseases affecting RPE. In conclusion, our protocol shortens the duration of isolation and simplifies the necessary techniques and reagents while maintaining high-purity cell culture.

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