Research reported in this publication was supported by NIH Grants R01EY018341 and R01EY019250&#160;(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.

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
<ol>
	<li>Prepare wash buffer medium by supplementing Hank&#39;s balanced salt solution (HBSS), no calcium, no magnesium, no phenol red with 10 mM HEPES buffer solution. Keep solution at 4 &#176;C until use.</li>
	<li>Prepare RPE medium by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium with 4.5 ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiol Rev. 85 (3), 845-881 (2005).</li><li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Prog Retin Eye Res. 100846, (2020).</li><li>Lambros, M. L., Plafker, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+the+Nrf2+anti-oxidant+transcription+factor+in+age-related+macular+degeneration.">Oxidative stress and the Nrf2 anti-oxidant transcription factor in age-related macular degeneration.</a> Adv Exp Med Biol. 854, 67-72 (2016).</li><li>Ferrari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa:+genes+and+disease+mechanisms.">Retinitis pigmentosa: genes and disease mechanisms.</a> Curr Genomics. 12 (4), 238-249 (2011).</li><li>Xia, T., Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+diabetic+retinopathy+on+the+barrier+functions+of+the+retinal+pigment+epithelium.">Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium.</a> Vision Res. 139, 72-81 (2017).</li><li>Edwards, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+rat+retinal+pigment+epithelium.">Culture of rat retinal pigment epithelium.</a> In Vitro. 13 (5), 301-304 (1977).</li><li>Mayerson, P. L., Hall, M. O., Clark, V., Abrams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+and+culture+of+rat+retinal+pigment+epithelial+cells.">An improved method for isolation and culture of rat retinal pigment epithelial cells.</a> Invest Ophthalmol Vis Sci. 26 (11), 1599-1609 (1985).</li><li>Chang, C. W., Roque, R. S., Defoe, D. M., Caldwell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+and+culture+of+pigment+epithelial+cells+from+rat+retina.">An improved method for isolation and culture of pigment epithelial cells from rat retina.</a> Curr Eye Res. 10 (11), 1081-1086 (1991).</li><li>Wang, N., Koutz, C. A., Anderson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+retinal+pigment+epithelial+cells+from+adult+rats.">A method for the isolation of retinal pigment epithelial cells from adult rats.</a> Invest Ophthalmol Vis Sci. 34 (1), 101-107 (1993).</li><li>Sakagami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+isolation+of+retinal+pigment+epithelial+cells+from+rat+eyeballs.">A rapid method for isolation of retinal pigment epithelial cells from rat eyeballs.</a> Ophthalmic Res. 27 (5), 262-267 (1995).</li><li>Heller, J. P., Kwok, J. C., Vecino, E., Martin, K. R., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+and+culture+of+adult+rat+retinal+pigment+epithelial+(RPE)+cells+to+study+retinal+diseases.">A method for the isolation and culture of adult rat retinal pigment epithelial (RPE) cells to study retinal diseases.</a> Front Cell Neurosci. 9, 449 (2015).</li><li>Shen, J., He, J., Wang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+retinal+pigment+epithelial+(RPE)+cells+with+Rho-Kinase+and+TGFbetaR-1/ALK5+inhibitor.">Isolation and culture of primary mouse retinal pigment epithelial (RPE) cells with Rho-Kinase and TGFbetaR-1/ALK5 inhibitor.</a> Med Sci Monit. 23, 6132-6136 (2017).</li><li>Hood, E. M. S., Curcio, C., Lipinski, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+cryosectioning+of+primary+porcine+retinal+pigment+epithelium+on+transwell+cell+culture+inserts.">Isolation, culture, and cryosectioning of primary porcine retinal pigment epithelium on transwell cell culture inserts.</a> STAR Protoc. 3 (4), 101758 (2022).</li><li>Naylor, A., Hopkins, A., Hudson, N., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+Junctions+of+the+outer+blood+retina+barrier.">Tight Junctions of the outer blood retina barrier.</a> Int J Mol Sci. 21 (1), 211 (2019).</li><li>Ban, B., Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+model+of+development+reveals+multiple+properties+of+RPE+tight+junctions.">A culture model of development reveals multiple properties of RPE tight junctions.</a> Mol Vis. 3, 18 (1997).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nat Protoc. 11 (7), 1206-1218 (2016).</li><li>Yang, S., Zhou, J., Dengwen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+diseases+of+the+retinal+pigment+epithelium.">Functions and diseases of the retinal pigment epithelium.</a> Front Pharmacol. 12, 727870 (2021).</li></ol>

There are no relevant financial or nonfinancial disclosures.

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...

The retinal pigment epithelium (RPE) is a specialized cell monolayer lining the Bruch&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.009 RD Single-Edge Blades</td>
			<td>Personna</td>
			<td>941202</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>with 4.5 g/L glucose, L-glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX, 100x</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>no Calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1M)</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids, 100x</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Unitome Knife</td>
			<td>BVI Beaver</td>
			<td>377546</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates</td>
			<td>Falcon</td>
			<td>08-772-1</td>
			<td>24-well or 48-well</td>
		</tr>
		<tr>
			<td>Regular Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>with phenol red</td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>10091-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified method for isolation and culture of retinal pigment epithelial cells from adult mice

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.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

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

Retinal pigment epithelial cells or RPE are located between photoreceptors and the choroid. Dysfunction of RPE is important in the pathogenesis of diseases, such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa. The use of primary RPE is key to studies to improve the understanding of how disease is developed.While various species have been used as a source of primary RPE, mice have the advantage of allowing to use genetic manipulation to help understand how retinopathies develop. Prior methods of isolation of primary RPE from rodents require either the use of neonatal animals, are lengthy, require technical expertise, or are not suitable for cell culture. We describe a simple and fast method to isolate primary RPE from adult mice that yields highly pure cultures of the cells.

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...

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.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

a-simplified-method-for-isolation-culture-retinal-pigment-epithelial

Research

JoVE Journal

Neuroscience

Extraction and Cultivation of Retinal Pigment Epithelial Cells from Adult Mice Eyes

To begin, prepare the wash buffer by supplementing Hank's Balanced Salt Solution with 10 millimolar heaps buffer. Store the buffer at four degrees Celsius until use. Prepare retinal pigment epithelium or RPE medium in DMEM and prewarm to 37 degrees Celsius in a water bath.Lay the euthanized mouse flat on its side with the eye facing upwards. Place the index finger above the eye and the thumb below it. Apply gentle pressure on the bone structure around the eye to cause the eyeball to protrude.Insert the tip of slightly open scissors under the eye and gently rotate the wrist away from the eye 90 degrees until the eye detaches from the socket. Immediately place and roll the eye in 70%ethanol for five seconds. Then transfer the eye to a wash buffer kept on ice.Under a dissecting microscope, place the eye in a Petri dish filled with wash buffer and strips of soaked gauze. Stabilize the eye by holding the optic nerve with tweezers and using a surgical knife gently make an incision at the level of the Ora Serrata. Insert Vannas Scissors into the incision and cut around the circumference of the Ora Serrata until the anterior segment and vitreous are removed.Gently peel away the retina from the eye cup using tethered forceps without disturbing the RPE layer. With the help of scissors, remove the optic nerve and excess connective tissue from the eye cup without puncturing the eye cup. Transfer the eye cup to a 1.5 milliliter microcentrifuge tube containing one milliliter of 0.25%trypsin and 0.02%EDTA.incubate the eye cup in a 37 degrees Celsius water bath for 10 minutes. Every two minutes, remove the tube and firmly tap the bottom 40 times onto the countertop. After incubation, using AP-1000 pipette gently mix up and down three times to disrupt the eye cup.To neutralize the trypsin, immediately layer the detached RPE sheets, excluding the eye cup onto 0.5 milliliters of FBS in a 15 milliliter conical tube. Then dropwise, add RPE media onto the layers of FBS and RPE sheets to dilute the trypsin. Centrifuge the RPE at 340G for three minutes.Discard the supernatant and resuspend the cells in a suitable amount of RPE media for a 24 well plate. Incubate the RPE at 37 degrees Celsius with 5%carbon dioxide for three days. 24 hours after RPE isolation, a nucleate pigmented cells begin to settle without full adherence.Over the 48 to 72 hours, these cells attach and spread, retaining dark pigmentation in a transparent nucleus. After five days, RPE cells show increased co fluency in start forming cell to cell contacts shaping into a polarized monolayer with hexagonal structures. Isolated RPE showed purity as evidenced by the RPE specific marker and tight junction integrity after six days in culture.

528 Views.  Case Western Reserve University.  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.

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

Summary