Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Les auteurs aimeraient montrer leur gratitude et leur respect à tous les animaux qui apportent leurs cellules dans cette étude. Cette étude a été financée en partie par des subventions du National Key R&D Program of China (2019YFA0111200, Yi Liao & Yuan Gao et Grant nos. 2018YFA0107301, Wei Li). Les auteurs remercient Jingru Huang et Xiang You du laboratoire central de la faculté de médecine de l’Université de Xiamen pour leur soutien technique en imagerie confocale.

L’utilisation d’animaux de laboratoire était conforme aux règlements de l’Association pour la recherche en vision et ophtalmologie (ARVO) et a été approuvée par le Comité d’éthique de la gestion des animaux expérimentaux de l’Université de Xiamen.
1. Préparation de dispositifs chirurgicaux expérimentaux, d’enzymes de digestion tissulaire et de tampons de culture cellulaire
<ol><li>Préparer les dispositifs chirurgicaux expérimentaux, en nettoyant et e...

<ol>
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S., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelium+polarity+in+health+and+blinding+diseases.">Retinal pigment epithelium polarity in health and blinding diseases.</a> Current Opinion in Cell Biology. 62, 37-45 (2020).</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> Progress in Retinal and Eye Research. 78, 100846 (2020).</li><li>Somasundaran, S., Constable, I. J., Mellough, C. B., Carvalho, L. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanin+pigment+in+the+pigmented+epithelium+of+the+retina+of+the+embryo+chick+eye+studied+in+vivo+and+in+vino.">Melanin pigment in the pigmented epithelium of the retina of the embryo chick eye studied in vivo and in vino.</a> The Anatomical Record. 18, 260-261 (1920).</li><li>Schnichels, 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=Retina+in+a+dish:+Cell+cultures,+retinal+explants+and+animal+models+for+common+diseases+of+the+retina.">Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.</a> Progress in Retinal and Eye Research. 81, 100880 (2021).</li><li>D'Antonio-Chronowska, A., D'Antonio, M., Frazer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+iPSC-derived+retinal+pigment+epithelium+cells+(iPSC-RPE).">In vitro differentiation of human iPSC-derived retinal pigment epithelium cells (iPSC-RPE).</a> Bio-Protocol. 9 (24), 3469 (2019).</li><li>Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+of+the+human+RPE+cell+line,+ARPE-19,+induced+by+nicotinamide.">Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.</a> Experimental Eye Research. 179, 18-24 (2019).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>. Scientific, T Available from: <a target="_blank" href="https://www.thermofisher.cn/document-connect/document">https://www.thermofisher.cn/document-connect/document</a> (2022)</li><li>. Toyota Available from: <a target="_blank" href="https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf">https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf</a> (2022)</li><li>. Abcam Available from: <a target="_blank" href="https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol">https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol</a> (2022)</li><li>. Zeiss Available from: <a target="_blank" href="https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals">https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals</a> (2022)</li><li>. Cell Signal Technology Available from: <a target="_blank" href="https://www.cellsignal.cn/learn-and-support/protocols/protocol-western">https://www.cellsignal.cn/learn-and-support/protocols/protocol-western</a> (2022)</li><li>Wang, 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=Reversed+senescence+of+retinal+pigment+epithelial+cell+by+coculture+with+embryonic+stem+cell+via+the+TGFbeta+and+PI3K+pathways.">Reversed senescence of retinal pigment epithelial cell by coculture with embryonic stem cell via the TGFbeta and PI3K pathways.</a> Frontiers in Cell and Developmental Biology. 8, 588050 (2020).</li><li>Pfeffer, B. A., Philp, N. 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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> Nature Protocols. 11 (7), 1206-1218 (2016).</li><li>Langenfeld, A., Julien, S., Schraermeyer, U. <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+the+isolation+and+culture+of+retinal+pigment+epithelial+cells+from+adult+rats.">An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253 (9), 1493-1502 (2015).</li><li>Sonoda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nature Protocols. 4 (5), 662-673 (2009).</li></ol>

Ici, un protocole détaillé et optimisé pour l’isolement, la culture et la caractérisation des cellules EPR à partir de globes oculaires porcins, qui génère un bon modèle pour la caractérisation in vitro des cellules EPR et les études sur les troubles liés à l’EPR, a été décrit. Des méthodes d’isolement de l’EPR à partir d’yeux humains, de souris et de rats ont été décrites précédemment23,24,25<...

L’épithélium pigmentaire rétinien (EPR) est situé entre les photorécepteurs et la choriocapillarise dans la couche externe de la rétine1 avec de multiples fonctions, y compris la formation de la barrière hémato-rétinienne, le transport et l’échange de nutriments et de métabolites rétiniens, le recyclage de la vitamine A pour maintenir un cycle visuel normal, et la phagocytose et la clairance des segments externes des photorécepteurs (POS)2,3 . Étant donné que les PDV nécessitent un auto-renouvellement constant pour générer la vision, les cellules EPR doivent continuellement engloutir les POS détachés pour maintenir l’homéostasie rétinienne4. Par conséquent, le dysfonctionnement de l’EPR entraîne de nombreuses maladies oculaires cécitantes, telles que la dégénérescence maculaire liée à l’âge (DMLA)4, la rétinite pigmentaire (RP)5, l’amaurose congénitale de Leber6, la rétinopathie diabétique7, etc. Jusqu’à présent, la pathogenèse exacte de la plupart de ces maladies reste insaisissable. En conséquence, la culture cellulaire RPE est établie pour étudier la biologie cellulaire RPE, les changements pathologiques et les mécanismes sous-jacents.
En tant que modèle le plus simple pour étudier la biologie cellulaire, la culture de cellules EPR a commencé dès les années 19208. Bien que l’ARPE-19 soit largement utilisé comme cellules EPR, la perte de pigmentation, la morphologie des pavés et, surtout, les fonctions de barrière dans cette lignée cellulaire soulèvent beaucoup de préoccupations9. En comparaison, la culture de cellules EPR humaines primaires offre un scénario plus réaliste pour les études physiologiques et pathologiques9. Cependant, la disponibilité relativement limitée restreint leur utilisation et des problèmes éthiques existent toujours. En outre, plusieurs groupes ont utilisé des modèles murins pour cultiver des cellules EPR. Cependant, la taille de l’œil de souris est petite et une seule culture nécessite généralement de nombreuses souris, ce qui n’est pas pratique9. Récemment, les scientifiques ont mis au point de nouvelles méthodes pour utiliser des cellules souches embryonnaires humaines ou des cellules souches pluripotentes induites pour dériver des cellules EPR. Bien que cette technique ait un potentiel particulier pour le traitement des troubles héréditaires de l’EPR, elle prend beaucoup de temps et nécessite généralement plusieurs mois pour générer des cellules EPR matures10. Pour surmonter ces problèmes, nous introduisons ici un protocole facile à suivre pour isoler et cultiver régulièrement des cellules EPR de haute pureté en laboratoire. Dans des conditions de culture appropriées, ces cellules peuvent présenter des fonctions RPE typiques et présenter des morphologies RPE typiques. Par conséquent, cette méthode de culture peut fournir un bon modèle pour comprendre la physiologie de l’EPR, étudier la cytotoxicité, étudier les mécanismes pathologiques des maladies oculaires connexes et effectuer des dépistages de médicaments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

primary culture of porcine retinal pigment epithelial cells

L’épithélium pigmentaire rétinien (EPR) est une monocouche de cellules épithéliales pigmentées polarisées, située entre la choroïde et la neurorétine dans la rétine. De multiples fonctions, y compris la phagocytose, le transport des nutriments / métabolites, le métabolisme de la vitamine A, etc., sont effectuées quotidiennement par l’EPR. Les cellules EPR sont des cellules épithéliales différenciées en phase terminale avec peu ou pas de capacité de régénération. La perte de cellules EPR entraîne de multiples maladies oculaires entraînant une déficience visuelle, telles que la dégénérescence maculaire liée à l’âge. Par conséquent, l’établissement d’un modèle de culture in vitro de cellules EPR primaires, qui ressemble plus à l’EPR in vivo qu’aux lignées cellulaires, est essentiel pour les études caractéristiques et mécanistes des cellules EPR. Compte tenu du fait que la source des globes oculaires humains est limitée, nous créons un protocole pour cultiver des cellules primaires d’EPR porcines. En utilisant ce protocole, les cellules EPR peuvent être facilement dissociées des globes oculaires porcins adultes. Par la suite, ces cellules dissociées se fixent aux boîtes de culture, prolifèrent pour former une monocouche confluente et rétablissent rapidement les principales caractéristiques du tissu épithélial in vivo en 2 semaines. Par qRT-PCR, il est démontré que les cellules RPE porcines primaires expriment plusieurs gènes de signature à des niveaux comparables avec le tissu RPE natif, tandis que les expressions de la plupart de ces gènes sont perdues / fortement réduites dans les cellules humaines de type EPR, ARPE-19. De plus, la coloration par immunofluorescence montre la distribution de la jonction serrée, de la polarité tissulaire et des protéines du cytosquelette, ainsi que la présence de RPE65, une isomérase essentielle au métabolisme de la vitamine A, dans les cellules primaires en culture. Dans l’ensemble, nous avons développé une approche facile à suivre pour la culture de cellules EPR porcines primaires avec des caractéristiques RPE de haute pureté et natives, qui pourrait servir de bon modèle pour comprendre la physiologie de l’EPR, étudier les toxicités cellulaires et faciliter les dépistages de médicaments.

Les cellules primaires de l’EPR porcine (EPRP) ont été cultivées dans un milieu DMEM/Basic avec 10 % de FBS, et la morphologie cellulaire au microscope optique a été photographiée 2 jours (figure 2A), 6 jours (figure 2B) et 10 jours (figure 2C) après l’ensemencement. Après 1 semaine, une monocouche confluente de cellules pRPE pigmentées avec des morphologies pavées a été observée.
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Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

Ici, une méthode facile à suivre pour cultiver in vitro des cellules épithéliales pigmentaires primaires de la rétine porcine est présentée.

Culture primaire de cellules épithéliales pigmentaires rétiniennes porcines

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Les troubles liés à l’EPR restent une partie importante du mélange des maladies, mais sans restes efficaces dans la culture virtuelle de cellules primaires de l’EPR pour servir de bon modèle pour les études physiologiques et pathologiques des troubles liés à l’EPR. Dans ce protocole, nous fournissons un protocole facile à suivre pour la culture de CPI primaires, qui pourrait rapidement restaurer et maintenir les caractéristiques RP ultérieures, nous croyons qu’en utilisant cette méthode, les gens pourraient rapidement générer des cellules RP à partir d’études de macistate et de criblages de médicaments protecteurs qui pourraient faciliter le développement de nouveaux traitements pour les troubles RPE. Une démonstration étape par étape de la valeur rendra cette méthode beaucoup plus facile à reproduire dans différents laboratoires qui souhaitent étudier les cellules RP.Pour commencer à décongeler l’enzyme de digestion tissulaire malaquad et stériliser 1X PBS. Complété par 2% de pénicilline et 2% de streptomycine en filtrant la solution à travers une unité filtrante à seringue de 0,22 micromètre. Lavez les puits de la plaque de culture et des inserts transwell avec 1X PBS.Retirez le PBS des puits et des puits avec une pipette et ajoutez un millilitre de PBS 1X frais dans la chambre inférieure et 600 microlitres de solution de laminine de 10 microgrammes par millilitre dans la chambre supérieure. Incuber les plaques et les inserts transwell dans cet incubateur hélicicole pendant la nuit à 37 degrés Celsius et 5% de dioxyde de carbone. Après cela, retirez la solution stratifiée de la chambre supérieure et lavez deux fois avec un millilitre de PBS 1X froid avant d’asseoir la cellule Nettoyez la hotte d’écoulement du stratifié avec de l’éthanol à 75% et de la lumière UV.Préparez trois tubes de centrifugation stériles de 50 millilitres contenant environ 25 millilitres d’éthanol à 75%, trois tubes de centrifugation stériles de 50 millilitres contenant environ 25 millilitres de 1X PBS et trois boîtes de culture cellulaire stérile de 10 centimètres contenant environ 15 millilitres de 1X PBS. Trempez quatre globes oculaires porcins dans environ 15 millilitres d’éthanol à 75% dans une boîte de Petri de 10 centimètres et utilisez une paire de ciseaux et de pinces pour couper tous les tissus conjonctifs et les muscles résiduels. Pour décontaminer les globes oculaires, faites tremper et lavez quatre globes oculaires dans trois tubes centrifugés stériles de 50 millilitres remplis séquentiellement d’éthanol à 75%.Ensuite, lavez les globes oculaires dans trois tubes centrifugés stériles de 50 millilitres remplis de 1X PBS séquentiellement. Inversez les tubes toutes les minutes pour bien laver les globes oculaires. Déplacez les quatre globes oculaires dans une boîte de culture cellulaire stérile de 10 centimètres contenant 1X PBS.Coupez à nouveau la surface externe de chaque globe oculaire pour enlever le nerf optique et les petits débris. Utilisez des ciseaux pour faire une petite coupure à l’intersection du limbe et de la sclérotique. Et puis retirez la cornée, l’iris, le cristallin, le corps vitré et la rétine neurale.Transférer le complexe de sclérotique choroïde RPE dans une nouvelle boîte de culture cellulaire de 10 centimètres et faire quatre coupes pour aplatir la coupelle oculaire en forme de trèfle à quatre feuilles. Effectuer des voyages dans la digestion de la solution d’EDTA en plaçant les quatre complexes de sclérotique choroïde RPE dans un nouveau plat de 10 centimètres et en versant 20 millilitres de solution d’EDTA de trypsine fraîche pour fusionner les complexes de sclérotique choroïde RPE. Placez le plat dans l’incubateur de culture cellulaire à 37 degrés Celsius pendant près de 30 minutes.Après 30 minutes, sortez la capsule de l’incubateur et ajoutez 20 millilitres de milieux de culture cellulaire préchaudes avec 10% FBS dans la boîte. Pour neutraliser les déclenchements en solution d’EDTA, utilisez une pipette de transfert de cinq millilitres pour dissocier les cellules EPR en pipetant doucement plusieurs fois. Recueillir les suspensions cellulaires dans des tubes centrifuges de 15 millilitres, puis pipeter doucement 10 millilitres supplémentaires de milieux de culture frais avec FBS pour laver les complexes de sclérotique RPE Choroids sur le plat.Recueillir les cellules EPR en centrifugeant les tubes à 300 G pendant cinq minutes à température ambiante. Aspirer le SuperAgent à l’aide d’une pipette et ajouter cinq millilitres supplémentaires de milieux de culture avec FBS pour remettre les cellules en suspension. Centrifuger à 300 G pendant encore cinq minutes.Décanter le SuperAgent et remettre les cellules en suspension avec 12 millilitres de milieux de culture. Prochaine graine de 100 000 à 200 000 cellules par puits dans 12 plaques de culture de puits ou inserts transwell. Changez les milieux de culture tous les deux jours et réduisez la concentration sérique à 1% lorsque les cellules couvrent complètement la surface des puits par insert.Changez les milieux de culture tous les deux jours jusqu’à ce que les cellules soient récoltées. Les images représentatives des cellules EPR porcines primaires du deuxième jour, du sixième jour et du jour 10 sont présentées ici. L’analyse QRTPCR des niveaux d’ARNm des gènes de signature dans les cellules RPE porcines primaires, les cellules RPE humaines primaires, les cellules ARPE 19 et le tissu choroïde RPE porcin est détectée par cette image graphique.Ici, GAP DH a été utilisé comme gène d’entretien pour RTPCR. Quatre réplicas biologiques ont été utilisés pour les cellules EPR porcines primaires et une cellule EPR 19 où trois réplicas biologiques ont été utilisés pour les cellules EPR humaines primaires et le tissu choroïde EPR porcin. Les niveaux d’expression génique dans les cellules ARPE 19 ont été définis comme témoins.L’analyse sanguine occidentale des protéines RPE 65 dans les cellules ARPE 19 et porcines RPE et dans les cellules RPE humaines primaires et les tissus choroïdes RPE porcins est illustrée dans cette figure. Le vinculin était utilisé ici comme contrôle de chargement. Cette figure montre une culture de deux semaines de cellules EPR porcines dans un milieu DMEM Basic avec 1% FBS.Les cellules cultivées avec ou sans Lamin ont été colorées avec RPE 65, APTA sodium-potassium, Z01 et Hex. Les mesures TR dans des feuilles cellulaires, cultivées avec ou sans laminine sont représentées dans cette figure. L’analyse sanguine occidentale du facteur de croissance de l’endothélium vasculaire ou VEGF et du facteur dérivé de l’épithélium pigmentaire ou PEDF dans les cellules RPE porcines primaires et ARPE 19 est illustrée dans cette figure.Les fonctions sectorielles des cellules RPE porcines primaires en culture et ARPE 19 sont présentées dans ces images. La grande partie de ce protocole consiste à dissoudre les cellules RP des complexes gliaux chides RPE. Nous recommandons d’utiliser des voyages frais et une solution à chaque fois et de contrôler correctement le temps de digestion, pour dissoudre huit cellules RP.

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Recherche

Biologie

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Profils MicroRNA d&#39;expression de cellules iPS humaines, l&#39;épithélium pigmentaire rétinien Derived From iPS, et fœtale épithélium pigmentaire rétinien

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Sous-rétinienne Transplantation de cellules souches embryonnaires humaines pigmentaires rétiniennes dérivées de cellules épithéliales dans un modèle grand aux yeux de l’atrophie géographique

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Cultures de cellules primaires de l’épithélium pigmentaire rétinien de souris

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Isolement, culture et génie génétique de cellules épithéliales pigmentaires primaires de mammifères pour la thérapie génique non virale

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissection efficace et culture des cellules épithéliales rétiniennes primaires de pigment de souris

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolement de cellules épithéliales pigmentées rétiniennes primaires de souris

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Analyse en temps réel de la bioénergétique dans les cellules épithéliales pigmentaires rétiniennes humaines primaires à l’aide de la respirométrie à haute résolution

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

Isolement de cellules épithéliales pigmentaires rétiniennes à partir d’yeux de cobaye

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Protocole pour l’isolement des cellules épithéliales mammaires dans les tissus humains

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Pleins feux sur l’auteur : Intégration de la médecine orientale et occidentale pour le traitement de la mammite granulomateuse

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolement et culture de cellules épithéliales mammaires humaines primaires

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...