Este estudio fue financiado en parte por The BrightFocus Foundation (para DS).

Procedimientos que involucran animales sujetos han sido aprobados por el cuidado institucional del Animal y el Comité uso (IACUC) en Universidad de Pittsburgh
1. Preparar soluciones
<ol><li>Preparar medio de cultivo por suplir Dulbecco modificado Eagle Medium (DMEM), glucosa alta con 10% suero bovino fetal (FBS), 1% de penicilina/estreptomicina, 2,5 mM L-glutamina y aminoácidos no esenciales de 1 MEM x. Precaliente los medios en la incubadora de 37 ° C antes de usar.</li>
	<li>Preparar solución de trabajo de 2% (peso/vol) dispase II:<ol>
<li>Preparar una solución stock de dispase II de 100 mg/mL disolver 30 mg de dispase II en 300 μL tampón HEPES solución salina (50 mM HEPES KOH de pH 7.4, 150 mM NaCl). Este volumen es de 3 a 4 ratones, puede ser escalado para arriba o abajo basado en el número de ratones para el experimento). Esta acción puede guardarse durante al menos 1 semana a 4 ° C.</li>
		<li>Preparar la solución de trabajo de 2% dispase agregando 300 μL de 100 mg/mL valores II dispase a 1,2 mL de DMEM estéril, glucosa alta y la solución de filtración con filtro 0,22 μm. Este paso debe realizarse en el gabinete bajo condiciones estériles de flujo laminar. Caliente previamente el preparado dispase II en incubadora de 37 ° C antes de usar.</li>
	</ol>
</li>
</ol>2. obtención de los ojos de ratón
<ol><li>Eutanasia el ratón utilizando cualquier método aprobado y colóquela sobre una almohadilla absorbente.</li>
	<li>Guantes, coloque el pulgar y el dedo índice alrededor del ojo y presione suavemente la piel para proptose del globo ocular.</li>
	<li>Inserte la punta de la tijera angulada entre la piel y el globo ocular y corte cuidadosamente el ojo. Para evitar la contaminación, sumergir brevemente el ojo en etanol al 70%.</li>
	<li>Inmediatamente colocar el ojo en el tampón fosfato salino (PBS) en una placa Petri.</li>
	<li>Repita los pasos 2.2-2.4 para quitar el segundo ojo.</li>
</ol>3. disección de ojo
<ol><li>Retire con cuidado el tejido conectivo del ojo con el microscopio de disección. Esto puede hacerse en PBS o en la caja de Petri seca.<ol>
<li>Utilice pinzas de sutura para levantar los tejidos conectivos del globo ocular y recorto con unas tijeras de Vannas. No corte la esclera debido a que es prácticamente imposible obtener las ojeras posteriores intactas si se corta la esclera.</li>
		<li>Después de limpiar el tejido conectivo, mantener los globos oculares resultantes en PBS para el siguiente paso de lavado. Nota: Los siguientes pasos deben realizarse en el gabinete bajo condiciones estériles de flujo laminar.</li>
	</ol>
</li>
	<li>Lavar los ojos dos veces en precalentado a 37 ° C DMEM (glucosa alta). Cuidadosamente transfiera los globos oculares en el C ° 37 con DMEM (glucosa alta) con unas pinzas. Aspire los ojos medio y la transferencia a medio fresco en un plato nuevo.</li>
	<li>Aspirar el medio DMEM (glucosa alta) e incubar los ojos en la solución de trabajo de II dispase 2% (peso/vol) precalentado por 45 min en la incubadora de 37 ° C. Nota: En todos los pasos siguientes, colocar y manipular ojos u ojeras en el medio de crecimiento, que protegen el tejido y proporcionan un buen medio para la disección.</li>
	<li>Retirar la solución II dispase y lavar los ojos dos veces en un medio de aspirar el medio antiguo y agregando el nuevo medio de crecimiento con 37 ° C (Figura 1a). Nota: Después de disociación por dispase II, los globos oculares se convierten en suaves.</li>
	<li>Sujete el ojo con pinzas y haga una incisión alrededor de la ora serrata, de cada ojo con unas tijeras de Vannas bajo el microscopio de disección coloca en el flujo laminar gabinete.<ol>
<li>Mantener el globo ocular en la solución, retire con cuidado la córnea anterior ampliando el corte alrededor de la circunferencia de la serrata de ora y tirando la córnea anterior después de que la corte ha sido completado.</li>
		<li>Retire la cápsula de lente y el epitelio asociado iris pigmentado tirando suavemente de la ojera con pinza dentada (Figura 1b, 1C).</li>
	</ol>
</li>
	<li>Incubar las ojeras posteriores resultantes en el medio de crecimiento por 20 min a 37 ° C para facilitar la separación de la retina neural del RPE.</li>
	<li>El cilindro posterior a cortar 4 pétalos con el microscopio de disección colocada en el flujo laminar gabinete. Los cortes deben ser lo suficientemente largo para aplanar la ojera, pero suficientemente corto como para mantener los pétalos conectados (figura 1 d).<ol>
<li>Aplane el cilindro y extraiga la retina neural tirando muy suavemente con unas pinzas mientras mantiene el resto de complejo de RPE-coroides esclerótica con otro fórceps (Figura 1e). Empezar a tirar de los bordes en lugar del centro.</li>
	</ol>
</li>
	<li>Transferencia de todos los tejidos en cuartos en una nueva placa de cultivo estéril con medio de cultivo con 37 ° C.</li>
</ol>4. aislamiento de las células RPE primarias
<ol><li>Sosteniendo un pétalo del complejo EPR-coroides esclerótica en cuartos con pinzas de la multa estupenda, retire suavemente las hojas intactas de RPE de la membrana basal subyacente (membrana de Bruch) utilizando un cuchillo media luna microquirúrgica con el microscopio de disección colocado en el flujo laminar del gabinete (figura 1f).<ol>
<li>Transferencia de las hojas RPE en el nuevo plato de cultivo estériles conteniendo medio de cultivo utilizando una micropipeta P200. Remisión de hojas RPE, mojar las puntas de pipetas con medio de cultivo mediante pipeteo varias veces para evitar que el tejido se pegue dentro de la punta. RPE es claramente distinguible de la coroides: RPE parece más pardusco, mientras que la coroides es más oscuro y parece pegajosa y esponjosa.</li>
		<li>También utilizar la digestión enzimática y disociación suave sin fórceps para separar el RPE de la coroides14.</li>
	</ol>
</li>
	<li>Lavar las hojas RPE 2 - 3 veces usando precalentado 37 ° C medio de cultivo en la placa de cultivo estéril. Después de cada paso de lavado, recoger cuidadosamente las hojas RPE evitando otros tejidos tales como desechos de retina o coroides. Al final del último lavado recoger las hojas RPE con una micropipeta de P200 y colocarlos en un nuevo tubo de microcentrífuga de 1,5 mL. Nota: No es necesario centrifugar las hojas RPE: van sedimentos por gravedad dentro de 1 minuto.</li>
	<li>Retire el medio de crecimiento extra usando una micropipeta P200. Resuspender las células en medio fresco precalentado y triturate suavemente utilizando una micropipeta P200. Evitar formación de burbujas. Una suspensión unicelular es ideal, pero también evitar demasiado trituración, de lo contrario las células RPE no son capaces de sobrevivir. Recomendamos muy suavemente trituración para 40 - 50 veces.</li>
</ol>5. cultivo de células RPE
<ol><li>Las células en una placa de cultivo de la placa.<ol>
<li>Si aplicaciones posteriores de las células RPE requieren retención de su polaridad, placa RPE células de 2 ratones en una inserción de membrana poliéster 12 mm cargado en placas 12-bien. Las celdas de alta densidad de la galjanoplastia es deseada porque en ese caso las células RPE son capaces de mantener las propiedades originales, como el hexagonal de la forma y la pigmentación.</li>
	</ol>
</li>
	<li>No mover la placa o cambiar el medio de crecimiento de las primeras 72 h de cultivo. Después de 3 días, reemplazar el viejo medio con medio fresco crecimiento precalentado. Después de cambia el medio de cultivo cada dos días. Una vez las células RPE confluency, reducir la FBS en medio del crecimiento a 2%. Nota: Las células se pueden dividir con tripsina 0.25%. Sin embargo, después de dividir las células pueden perder su forma hexagonal y pigmentación en los pasos posteriores. No se recomienda dividir las células, pero si es necesaria para las aplicaciones posteriores de la cultura, tenga en cuenta que las células primarias no pueden ser pasadas por tiempo indefinido: se sabe que se distinguen después de 5 a 7 pasos.</li>
</ol>

<ol>
	<li>Mart&#237;nez-Morales, J. R., Rodrigo, I., Bovolenta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+development:+a+view+from+the+retina+pigmented+epithelium.">Eye development: a view from the retina pigmented epithelium.</a> Bioessays. 26, 766-777 (2004).</li><li>Lehmann, G. L., Benedicto, I., Philp, N. J., 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=Plasma+membrane+protein+polarity+and+trafficking+in+RPE+cells:+past,+present+and+future.">Plasma membrane protein polarity and trafficking in RPE cells: past, present and future.</a> Exp Eye Res. 126, 5-15 (2014).</li><li>Fronk, A. H., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+retinal+pigment+epithelial+cells:+a+review+of+current+protocols+and+future+recommendations.">Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.</a> J Tissue Eng. 7, (2016).</li><li>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=Barrier+properties+of+cultured+retinal+pigment+epithelium.">Barrier properties of cultured retinal pigment epithelium.</a> Exp Eye Res. 126, 16-26 (2014).</li><li>Lu, F., Yan, D., Zhou, X., Hu, D. N., Qu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+melanin-related+genes+in+cultured+adult+human+retinal+pigment+epithelium+and+uveal+melanoma+cells.">Expression of melanin-related genes in cultured adult human retinal pigment epithelium and uveal melanoma cells.</a> Mol Vis. 13, 2066-2072 (2007).</li><li>Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+melanin+and+lipofuscin+in+RPE+cell+culture+models.">Studying melanin and lipofuscin in RPE cell culture models.</a> Exp Eye Res. 126, 61-67 (2014).</li><li>Saini, J. S., Temple, S., Stern, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Retinal+Pigment+Epithelium+Stem+Cell+(RPESC).">Human Retinal Pigment Epithelium Stem Cell (RPESC).</a> Adv Exp Med Biol. 854, 557-562 (2016).</li><li>Akrami, H., 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=Retinal+pigment+epithelium+culture;a+potential+source+of+retinal+stem+cells.">Retinal pigment epithelium culture;a potential source of retinal stem cells.</a> J Ophthalmic Vis Res. 4, 134-141 (2009).</li><li>Hu, J., Bok, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cultured+human+fetal+retinal+pigment+epithelium+in+studies+of+the+classical+retinoid+visual+cycle+and+retinoid-based+disease+processes.">The use of cultured human fetal retinal pigment epithelium in studies of the classical retinoid visual cycle and retinoid-based disease processes.</a> Exp Eye Res. , 46-50 (2014).</li><li>Mazzoni, F., Safa, H., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+photoreceptor+outer+segment+phagocytosis:+use+and+utility+of+RPE+cells+in+culture.">Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.</a> Exp Eye Res. 126, 51-60 (2014).</li><li>Reichhart, N., 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=Ion+channels+and+transporters+of+the+retinal+pigment+epithelium.">Ion channels and transporters of the retinal pigment epithelium.</a> Exp Eye Res. 126, 27-37 (2014).</li><li>Valapala, M., 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=Lysosomal-mediated+waste+clearance+in+retinal+pigment+epithelial+cells+is+regulated+by+CRYBA1/&#946;A3/A1-crystallin+via+V-ATPase-MTORC1+signaling.">Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/&#946;A3/A1-crystallin via V-ATPase-MTORC1 signaling.</a> Autophagy. 10, 480-496 (2014).</li><li>Shang, P., 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+amino+acid+transporter+SLC36A4+regulates+the+amino+acid+pool+in+retinal+pigmented+epithelial+cells+and+mediates+the+mechanistic+target+of+rapamycin,+complex+1+signaling.">The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.</a> Aging Cell. , (2017).</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, 1206-1218 (2016).</li><li>Pfeffer, B. A., Philp, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+retinal+pigment+epithelium:+Special+Issue.">Cell culture of retinal pigment epithelium: Special Issue.</a> Exp Eye Res. 126, 1-4 (2014).</li><li>Singh, S., Woerly, S., McLaughlin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+artificial+substrates+for+retinal+pigment+epithelial+monolayer+transplantation.">Natural and artificial substrates for retinal pigment epithelial monolayer transplantation.</a> Biomaterials. 22, 3337-3343 (2001).</li><li>Sonoda, 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=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> Nat Protoc. 4, 662-673 (2009).</li><li>Ho, T. C., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+of+retinal+pigment+epithelium+following+isolation+with+a+gelatin+matrix+technique.">Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique.</a> Experimental eye research. 64, 133-139 (1997).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Exp Eye Res. , 74-85 (2014).</li><li>Gibbs, D., Kitamoto, J., 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=Abnormal+phagocytosis+by+retinal+pigmented+epithelium+that+lacks+myosin+VIIa,+the+Usher+syndrome+1B+protein.">Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.</a> Proc Natl Acad Sci USA. 100, 6481-6486 (2003).</li><li>Gibbs, D., 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=Isolation+and+culture+of+primary+mouse+retinal+pigmented+epithelial+cells.">Isolation and culture of primary mouse retinal pigmented epithelial cells.</a> Adv Exp Med Biol. 533, 347-352 (2003).</li><li>Pitk&#228;nen, L., Ranta, V. P., Moilanen, H., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+retinal+pigment+epithelium:+effects+of+permeant+molecular+weight+and+lipophilicity.">Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity.</a> Invest Ophthalmol Vis Sci. 46, 641-646 (2005).</li><li>Mao, Y., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+photoreceptor+outer+segment+phagocytosis+by+RPE+cells+in+culture.">Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture.</a> Methods Mol Biol. 935, 285-295 (2013).</li></ol>

DS ha recibido financiación de Alemania y F. Hoffmann-La Roche, Bayer HealthCare, Suiza de la investigación. Los restantes autores no declaran a intereses financieros que compiten. Este trabajo es apoyado por la investigación para evitar la ceguera (becas sin restricciones el Wilmer Eye Institute y la Universidad de Pittsburgh).  Este trabajo es apoyado también por los fondos de arranque para DS de Oftalmología, Universidad de Pittsburgh.

El protocolo detallado presentado permite confiable establecimiento de cultivos primarios RPE ratón que alcanzan confluencia después de 1 semana y presentan las principales características RPE como forma hexagonal y pigmentación después de 2 semanas. Las células RPE obtenidas pueden ser utilizadas para un número de aplicaciones posteriores tales como vitamina A metabolismo9, fagocitosis de los segmentos externo de fotorreceptor10 y ion transporte11<...

El epitelio retiniano del pigmento (RPE) es una sola capa de células epiteliales polarizadas que se encuentra entre la retina neural y la coroides del ojo. La funcionalidad de las células RPE y la integridad de la monocapa RPE son críticos para la visión porque del RPE desempeña un papel importante en varios procesos como el mantenimiento de la barrera sangre-retiniana externa, transporte de agua e iones entre la retina y el absorción de la coroide, luz, protección contra el estrés oxidativo, el control del metabolismo de los retinoides y fagocitosis de los segmentos externos de los fotorreceptores1,2. La ubicación de la ERP en la parte posterior del ojo, así como su función de barrera evitando fármacos administrados sistémicamente de paso de la sangre al humor vítreo, dificultar estudiar la complejidad de la función la EPR en vivo. Así, hay una gran necesidad para el establecimiento de cultivos de células RPE para el estudio de las células RPE en un ambiente flexible, controlado3,4.
Existe un número de líneas establecidas de células RPE, proporciona una manera fácil y conveniente de obtención y almacenamiento de las células; sin embargo, las células los tienen algunas desventajas en comparación con células primarias2,3,4. En primer lugar, a menudo se caracterizan por cambios en la morfología de la célula. Por ejemplo, ninguna de las líneas celulares existentes fueron encontrada para ser conveniente para un estudio confiable de las propiedades de barrera RPE debido a la pérdida del fenotipo de la polaridad celular y desaparición parcial de ensambladuras apretadas4. Además de la pérdida de polaridad y conexiones adecuadas de célula a célula, las líneas de células RPE rápidamente pierden su pigmentación debido a la ausencia de las enzimas clave de la melanogénesis en el RPE adulto5. La pigmentación puede ser restaurada, pero el análisis integral del mecanismo de la pigmentación que incluiría una combinación de microscopía electrónica de transmisión, ensayos de química y análisis de expresión genética para confirmar la presencia de melanina nunca ha se han realizado6. Una limitación más es que las líneas de células RPE tienen vida extendido (a veces - la inmortalidad) y bajo ciertas condiciones pueden transformarse en renovado células madre multipotentes que separar del sustrato y forma flotante colonias7, 8. esta limitación hace que sea imposible utilizar las líneas de células para trasplante de experimentos3.
Teniendo en cuenta las desventajas de las líneas establecidas de células RPE, cultivos primarios de células RPE obtenidos de tejidos frescos podrían servir como un modelo biológicamente más relevante para el estudio del RPE. Las células RPE primarias han sido utilizadas no sólo para estudiar las funciones específicas de RPE como metabolismo de la vitamina A9, fagocitosis de los segmentos externo de fotorreceptor10 y ion transporte11, sino también para estudiar biología celular básica tales como epiteliales polaridad de la célula2 , homeostasis lisosomal y autofagia12,13.
En los últimos años ha habido un número de publicaciones sobre el establecimiento de cultivos primarios de RPE, que indica un creciente interés en esta área de investigación3,14,15. Numerosos protocolos para células RPE humanas y no humanas RPE células como las células RPE de bovina y porcinas fueron publicados16,17,18,19. Sin embargo, es más difícil manejar las células RPE ratón debido a su tamaño mucho más pequeño. A pesar de que absolutamente un número de publicaciones ha descrito protocolos para aislar células RPE del ratón14,20,21, todavía hay muchos investigadores que luchan aislar las células RPE sin contaminación de células coroides o de desechos de retina neural. Aquí presentamos el protocolo para el establecimiento de cultivo de células RPE, incluyendo la obtención de los ojos del ratón, la disección de los ojos y el aislamiento de las hojas RPE para producir las células de cultivo primario de ratón. Este protocolo video sería especialmente útil para los investigadores que están empezando a trabajar con cultivos primarios de RPE de ratón y necesita orientación sobre las técnicas de disección.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">animals</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">2~3-week old wildtype mice</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">reagents</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11965092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dispase II</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">D4693</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Phosphate-buffered saline (PBS), 1X, pH 7.4</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">HEPES</td>
			<td style="width:123px;">Cellgro</td>
			<td style="width:123px;">61-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">KOH</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">1310-58-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaCl</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:123px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">L-Glutamine (200 mM)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ethyl Alcohol</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">111000200</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:38px;width:347px;">RPE65 antibody</td>
			<td style="width:123px;">A gift from Dr. Michael Redmond</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fluorescein Phalloidin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">F432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat anti-Rabbit IgG (H+L), Alexa Flour 568&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">A11011&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat serum</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DAPI</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">Polysciences, Inc</td>
			<td style="width:123px;">00380</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:38px;width:347px;">ZO-1 Polyclonal Antibody</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:123px;">40-2200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">instruments and equipments</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Laminar flow cabinet</td>
			<td style="width:123px;">Baker</td>
			<td style="width:123px;">SterilGARD SG403A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dissecting microscope (Zoom stereomicroscope)</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:123px;">SMZ1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">CO2 incubator with hot air sterilization</td>
			<td style="width:123px;">Binder</td>
			<td style="width:123px;">C150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:123px;">5702</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Petri dishes</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">0875712</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">12 mm Polyester Membrane Inserts Pre-Loaded in 12-Well Culture Plates, Pore Size: 0.4 &#181;m, Sterile</td>
			<td style="width:123px;">Corning Incorporated</td>
			<td style="width:123px;">COR-3460</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Westcott tenotomy scissors, std blades, sharp</td>
			<td style="width:123px;">STEPHENS instruments</td>
			<td style="width:123px;">S7-1320</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Castroviejo suturing forceps 0.12mm</td>
			<td style="width:123px;">Stroz Ophthalmic Instruments</td>
			<td style="width:123px;">E1796</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Crescent straight knife</td>
			<td style="width:123px;">Beaver-Visitec International</td>
			<td style="width:123px;">373808</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Dumont Tweezers #5, 11 cm, Straight, 0.1x0.06 mm Tips, Dumostar</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500233</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Vannas Scissors, 8 cm, 45&#176; Angle, Standard</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Millex-GS Syringe Filter Unit, 0.22 &#181;m</td>
			<td style="width:123px;">Millipore</td>
			<td style="width:123px;">SLGL0250S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Syringe, 5 mL</td>
			<td style="width:123px;">BD</td>
			<td style="width:123px;">309632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Inverted Laboratory Microscope&#160;Leica DM IL LED</td>
			<td style="width:123px;">&#160;Leica&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipette</td>
			<td style="width:123px;">Gilson</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Barrier and non-filtered pipette tips</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

primary cell cultures from the mouse retinal pigment epithelium

El epitelio retiniano del pigmento (RPE) es un epitelio multifuncional altamente polarizado que se encuentra entre la retina neural y la coroides del ojo. Es una sola hoja de células pigmentadas que es hexagonal embalado y conectadas por uniones estrechas. Las principales funciones del RPE incluyen la absorción de la luz, fagocitosis de los segmentos externo del fotorreceptor de vertiente, búfer espacial de iones, transporte de nutrientes, iones y agua, así como participación activa en el ciclo visual. Con tales funciones importantes y diversas, es críticamente importante para el estudio de la biología de las células RPE. Se han establecido una serie de líneas de células RPE; sin embargo, las células inmortalizadas y los conocen a perder rápidamente algunas de las características morfológicas y fisiológicas de las células RPE naturales. Así, células primarias son más adecuadas para el estudio de diferentes aspectos de la biología de la célula RPE y función. Cultivo de células RPE primaria de ratón es muy útil a los investigadores desde modelos de ratón son ampliamente utilizados en estudios de biología, sin embargo recoger RPE células de ratón también es muy difícil debido a su pequeño tamaño. Aquí, presentamos un protocolo para el establecimiento de cultivos de células primarias de ratón RPE que incluye enucleación y disección de los ojos y el aislamiento de las hojas RPE para producir las células de cultivo. Este método permite la recuperación eficiente de la célula. Las células RPE de dos ratones pueden llegar a la confluencia en una inserción de membrana poliéster 12 mm cargado en placa de cultivo después de una semana de la cultura y mostrar algunas de las propiedades originales de buena fe RPE células como forma hexagonal y pigmentación después de dos semanas de la cultura.

El ratón RPE cultivo primario establecido de 2 ratones en 12 mm membrana poliéster relleno alcanza 90-95% confluencia después de 1 semana en cultura. Después de 2 semanas de cultivo, las células alcanzaron 100% de confluencia y comenzaron a formar un mosaico de células hexagonales pigmentadas y bi-nucleated. Por 3 semanas de cultivo, las células continúan forman la forma y la pigmentación, sin embargo, después de 4 semanas una porción de células consiguió hiperpigmentadas (<s...

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

El epitelio retiniano del pigmento (RPE) es un epitelio de múltiples funciones del ojo. Aquí presentamos un protocolo para establecer cultivos celulares primarios derivados del RPE murino.

Cultivos celulares primarios de células del epitelio pigmentario de la retina del ratón

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

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Research

JoVE Journal

Biology

20.9K vistas.  University of Pittsburgh School of Medicine. El epitelio retiniano del pigmento (RPE) es un epitelio de múltiples funciones del ojo. Aquí presentamos un protocolo para establecer cultivos celulares primarios derivados del RPE murino.

Video: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Cultivos neuronales primarios del cerebro de Drosophila pupas etapa tardía

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Investigación

Biología

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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.

Perfiles de expresión de microARN de las células iPS humanas, de la retina del epitelio pigmentario Derivado de Ips y Fetal epitelio pigmentario de la retina

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

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.

Disección eficiente y cultivo de células epiteliales de pigmento retiniano de ratón primario

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

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Ablación mediada por nitrorreductasa/metronidazol y una plataforma MATLAB (RpEGEN) para estudiar la regeneración del epitelio pigmentario de la retina del pez cebra

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

Cultivo primario de células epiteliales pigmentarias de la retina porcina

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

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.

Aislamiento de células primarias del epitelio pigmentado de la retina del ratón

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