Name: Författare Spotlight: Effektiv isolering av primära retinala pigmentepitelceller från vuxna möss för retinopatiforskning
Uploaded: 2024-05-24T14:50:00
Description: Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Forskning som rapporteras i denna publikation stöddes av NIH Grants R01EY018341 and R01EY019250 (C.S.S.), NIH Grant F31EY035156 (A.H.) och P30 EY011373. Finansiären hade ingen roll i utformningen eller genomförandet av denna forskning.

Användningen av försökspersoner i denna studie godkändes av Institutional Animal Care and Use Committee (IACUC) vid Case Western Reserve University.
1. Förberedelse av reagenser
<ol><li>Förbered tvättbuffertmediet genom att komplettera Hanks balanserade saltlösning (HBSS), inget kalcium, inget magnesium, inget fenolrött med 10 mM HEPES buffertlösning. Förvara lösningen vid 4 °C tills den ska användas.</li>
	<li>Förbered RPE-mediet genom att komplettera Dulbecc...

Karakterisering och morfologisk dynamik av odlad primär murin 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>

I den här artikeln har vi beskrivit ett förenklat protokoll för isolering och odling av murint retinalt pigmentepitel. RPE-celler isolerade från ögonen hos vuxna möss uttryckte en RPE-specifik markör, RPE65, och en intercellulär korsningsmarkör, ZO-1. Dessutom utvecklades de odlade cellerna till pigmenterade, sexkantiga ark i odling.
Flera metoder för isolering av RPE hos gnagare har tidigare publicerats 6,7,8,9,10,11,12.<sup...

Det retinala pigmentepitelet (RPE) är ett specialiserat cellmonolager som kantar Bruchs membran som ligger mellan fotoreceptorer och åderhinnan1. RPE-celler spelar en avgörande roll för att näthinnan ska fungera korrekt. RPE-celler transporterar glukos och vitamin A till fotoreceptorer, främjar synen genom omisomerisering av all-trans retinal till 11-cis retinal och upprätthåller yttre segment av fotoreceptorn genom fagocytos av utsöndrade yttre segment, avlägsnar vatten från det subretinala utrymmet, bildar den yttre blod-retinala barriären genom närvaron av täta korsningar och utsöndrar neurotropa tillväxtfaktorer (såsom Pigment Epithelium Derived Factor, och Basic Fibroblast Growth Factor) som stöder fotoreceptorer2. Dysfunktion av RPE-celler är involverad i patogenesen av olika retinopatier, inklusive åldersrelaterad makuladegeneration, retinitis pigmentosa och diabetisk retinopati 3,4,5. In vitro-studier med RPE-celler är avgörande för att förbättra vår förståelse av patogenesen av dessa sjukdomar. Primära RPE-celler är mycket att föredra för dessa studier eftersom RPE-cellinjer, även om de är lättillgängliga, saknar viktiga egenskaper hos primära RPE-celler.
Medan olika arter har använts som källor till primära RPE-celler, har möss fördelen att använda genetiska modifieringar för att hjälpa till att förstå patogenesen av retinopatier. Tidigare beskrivna protokoll för att isolera RPE-celler från gnagare kräver antingen användning av neonatala djur, är långa, kräver teknisk skicklighet eller är inte lämpliga för odling 6,7,8,9,10,11,12. Vi beskriver en enkel och snabb metod för att isolera RPE-celler från vuxna möss som ger mycket rena kulturer av dessa celler.

<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

Retinala pigmentepitelceller (RPE) är avgörande för att näthinnan ska fungera korrekt. RPE-dysfunktion är involverad i patogenesen av viktiga näthinnesjukdomar, såsom åldersrelaterad makuladegeneration, retinitis pigmentosa och diabetisk retinopati. Vi presenterar ett strömlinjeformat tillvägagångssätt för isolering av RPE från vuxna ögon hos möss. Till skillnad från tidigare rapporterade metoder möjliggör detta tillvägagångssätt isolering och odling av mycket ren RPE från vuxna möss. Denna enkla och snabba metod kräver ingen omfattande teknisk skicklighet och kan uppnås med grundläggande vetenskapliga verktyg och reagenser. Primär RPE isoleras från C57BL/6-bakgrundsmöss i åldern 3 till 14 veckor genom enukleation av ögat följt av avlägsnande av det främre segmentet. Enzymatisk trypsinisering och centrifugering används för att dissociera och isolera RPE från ögonmusslan. Sammanfattningsvis erbjuder detta tillvägagångssätt ett snabbt och effektivt protokoll för användning av RPE i studien av näthinnans funktion och sjukdom.

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.

Författare Spotlight: Effektiv isolering av primära retinala pigmentepitelceller från vuxna möss för retinopatiforskning

En förenklad metod för isolering och odling av retinala pigmentepitelceller från vuxna möss

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.

Det beskrivna protokollet har använts på C57BL/6 bakgrundsmöss. Kön verkar inte förändra förmågan att odla RPE. Möss under 6 veckor ger begränsade RPE-ark i jämförelse med äldre möss, och fler ögon kan behövas för att nå optimalt sammanflöde. Efter isolering tar det ungefär 3 dagar för RPE-celler att stabiliseras och fästa på cellodlingsplattan. Ungefär 24 timmar efter isolering har runda, pigmenterade celler som ser ut att vara anukleära börjat sätta sig men har inte helt fäst vid plattan (<s...

Retinalt pigmentepitel (RPE) fungerar som en avgörande barriär mellan åderhinnan och näthinnan och främjar hälsan och funktionen hos näthinnans celltyper, såsom fotoreceptorer. Här beskriver vi ett enkelt och effektivt protokoll för att isolera och odla vuxen murin 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

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.

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.

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.