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 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 &#176;C in a water bath.</li>
</ol>
2. Mouse eye extraction
<ol>
	<li>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.</li>
	<li>Lay the mouse flat on its side with the eye oriented upwards.</li>
	<li>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.</li>
	<li>Insert the tip of slightly opened scissors underneath the eye and gently rotate the wrist away from the eye 90&#176; 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.</li>
	<li>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.</li>
	<li>Under a dissecting microscope, transfer one eye at a time to a Petri dish filled with wash buffer and strips of soaked gauze.</li>
	<li>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&#176; surgical knife or 0.009&#34; razor blade.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Finally, remove the optic nerve and excess connective tissue from the eyecup using scissors. Be careful not to puncture the eyecup.</li>
</ol>
3. Isolation of primary RPE
<ol>
	<li>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.</li>
	<li>Transfer microcentrifuge tubes to a water bath set at 37 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Further, dilute the trypsin by layering 3 mL of RPE media dropwise onto the layers of FBS and RPE sheets.</li>
	<li>Centrifuge the RPE at 340 x g for 3 min.</li>
	<li>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.</li>
	<li>Incubate at 37 &#176;C at 5% CO2.</li>
</ol>
4. Culturing RPE
<ol>
	<li>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.</li>
	<li>Change the medium every 48 h after the first 3 days.</li>
	<li>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.</li>
</ol>

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 previously6,7,8,9,10,11,12. Many protocols peel the RPE layers from the Bruch&#39;s membrane requiring technical skill and increasing the risk of RPE damage or cell death6,7,9,11,12,16. 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&#39;s membrane. This protocol has been successfully performed on mice aged 3-&#160;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&#39;s membrane and choroid, increase incubation time with trypsin in the water bath. Additionally, freeze aliquots of fresh trypsin at -20 &#176;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 TGF&#946;R-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.

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66921/66921fig01.jpg" /> 
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)&#160;After 48 h and 72 h, primary RPE begin to adhere to the plate and form pigmented, binucleated cells. (D)&#160;After 5 days, brightfield microscopy shows the hyperpigmented, hexagonal-shaped cells increasing confluency and beginning to form cell-to-cell contacts. Scale bar: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66921/66921fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66921/66921fig02.jpg" /> 
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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66921/66921fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66921/66921fig03.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/66921/66921fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

900 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.

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...