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

Los autores desean mostrar su gratitud y respeto a todos los animales que contribuyen con sus células en este estudio. Este estudio fue apoyado en parte por subvenciones del Programa Nacional de Investigación y Desarrollo Clave de China (2019YFA0111200, Yi Liao & Yuan Gao y Grant nos. 2018YFA0107301, Wei Li). Los autores agradecen a Jingru Huang y Xiang You del Laboratorio Central, Facultad de Medicina de la Universidad de Xiamen por el apoyo técnico en imágenes confocales.

El uso de animales de experimentación cumplió con las regulaciones de la Asociación para la Investigación en Visión y Oftalmología (ARVO) y fue aprobado por el Comité de Ética de Manejo de Animales Experimentales de la Universidad de Xiamen.
1. Preparación de dispositivos quirúrgicos experimentales, enzima de digestión tisular y tampón de cultivo celular
<ol><li>Preparar los dispositivos quirúrgicos experimentales, limpiando y esterilizando en autoclave dos pare...

<ol>
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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>. 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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>

Aquí, se ha descrito un protocolo detallado y optimizado para el aislamiento, cultivo y caracterización de células de EPR de globos oculares porcinos, que genera un buen modelo para la caracterización in vitro de células de EPR y estudios de trastornos relacionados con EPR. Los métodos para el aislamiento del EPR de los ojos humanos, de ratón y de rata se han descrito previamente23,24,25. Sin embargo, es difícil...

El epitelio pigmentario de la retina (EPR) se encuentra entre los fotorreceptores y el coriocapilar en la capa externa de la retina1 con múltiples funciones, incluida la formación de la barrera hematoretiniana, el transporte e intercambio de nutrientes y metabolitos retinianos, el reciclaje de vitamina A para mantener un ciclo visual normal, y la fagocitosis y eliminación de los segmentos externos fotorreceptores (POS) desprendidos2,3 . Dado que los POS requieren una auto-renovación constante para generar visión, las células RPE necesitan engullir continuamente POS separados para mantener la homeostasis retiniana4. Por lo tanto, la disfunción del EPR da lugar a muchas enfermedades oculares cegadoras, como la degeneración macular asociada a la edad (DMAE)4, la retinitis pigmentosa (RP)5, la amaurosis congénita de Leber6, la retinopatía diabética7, etc. Hasta ahora, la patogénesis exacta de la mayoría de estas enfermedades sigue siendo difícil de alcanzar. Como resultado, el cultivo celular de EPR se establece para estudiar la biología celular del EPR, los cambios patológicos y los mecanismos subyacentes.
Como el modelo más simple para estudiar la biología celular, el cultivo de células RPE se inició ya en la década de 19208. Aunque ARPE-19 es ampliamente utilizado como células del EPR, la pérdida de pigmentación, la morfología de los adoquines y, especialmente, las funciones de barrera en esta línea celular plantean muchas preocupaciones9. En comparación, el cultivo de células RPE humanas primarias ofrece un escenario más realista para estudios fisiológicos y patológicos9. Sin embargo, la disponibilidad relativamente limitada restringe su uso y siempre existen problemas éticos. Además, varios grupos utilizaron modelos de ratón para cultivar células de EPR. Sin embargo, el tamaño del ojo del ratón es pequeño, y un solo cultivo generalmente requiere muchos ratones, lo cual no es conveniente9. Recientemente, los científicos han desarrollado nuevos métodos para utilizar células madre embrionarias humanas o células madre pluripotentes inducidas para derivar células RPE. Aunque esta técnica tiene un potencial particular para el tratamiento de trastornos hereditarios del EPR, consume mucho tiempo y generalmente requiere varios meses para generar células maduras del EPR10. Para superar estos problemas, aquí presentamos un protocolo fácil de seguir para aislar y cultivar células RPE de alta pureza en el laboratorio de forma rutinaria. En condiciones de cultivo adecuadas, estas células pueden mostrar funciones típicas del EPR y exhibir morfologías típicas del EPR. Por lo tanto, este método de cultivo puede proporcionar un buen modelo para comprender la fisiología del EPR, estudiar la citotoxicidad, investigar los mecanismos patológicos de las enfermedades oculares relacionadas y realizar exámenes de detección de drogas.

<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

El epitelio pigmentario de la retina (EPR) es una monocapa de células epiteliales pigmentadas polarizadas, ubicadas entre la coroides y la neuroretina en la retina. El RPE realiza diariamente múltiples funciones, incluyendo fagocitosis, transporte de nutrientes/metabolitos, metabolismo de la vitamina A, etc. Las células del EPR son células epiteliales terminalmente diferenciadas con poca o ninguna capacidad regenerativa. La pérdida de células del EPR da lugar a múltiples enfermedades oculares que conducen a la discapacidad visual, como la degeneración macular relacionada con la edad. Por lo tanto, el establecimiento de un modelo de cultivo in vitro de células primarias del EPR, que se asemeje más al EPR in vivo que a las líneas celulares, es fundamental para los estudios característicos y mecanicistas de las células del EPR. Teniendo en cuenta el hecho de que la fuente de globos oculares humanos es limitada, creamos un protocolo para cultivar células primarias de EPR porcino. Mediante el uso de este protocolo, las células RPE se pueden disociar fácilmente de los globos oculares porcinos adultos. Posteriormente, estas células disociadas se adhieren a placas / inserciones de cultivo, proliferan para formar una monocapa confluente y restablecen rápidamente las características clave del tejido epitelial in vivo dentro de las 2 semanas. Mediante qRT-PCR, se demuestra que las células primarias del EPR porcino expresan múltiples genes de firma a niveles comparables con el tejido RPE nativo, mientras que las expresiones de la mayoría de estos genes se pierden/se reducen altamente en las células humanas similares al EPR, ARPE-19. Además, la tinción de inmunofluorescencia muestra la distribución de la unión estrecha, la polaridad tisular y las proteínas del citoesqueleto, así como la presencia de RPE65, una isomerasa crítica para el metabolismo de la vitamina A, en células primarias cultivadas. En conjunto, hemos desarrollado un enfoque fácil de seguir para cultivar células primarias de EPR porcino con alta pureza y características nativas de EPR, que podría servir como un buen modelo para comprender la fisiología del EPR, estudiar las toxicidades celulares y facilitar las pruebas de detección de fármacos.

Las células primarias de EPR porcino (pRPE) se cultivaron en medios DMEM/Basic con 10% de FBS, y la morfología celular bajo microscopio óptico se fotografió a los 2 días (Figura 2A), 6 días (Figura 2B) y 10 días (Figura 2C) después de la siembra. Después de 1 semana, se observó una monocapa confluente de células pRPE pigmentadas con morfologías de adoquines.
Para caracterizar mejor las célula...

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

Aquí, se presenta un método fácil de seguir para cultivar células epiteliales pigmentarias primarias de la retina porcina in vitro .

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. ...

Los trastornos relacionados con el EPR siguen siendo una parte importante de la mezcla de enfermedades, pero sin restos efectivos en el cultivo virtual de células primarias del EPR que sirvan como un buen modelo para los estudios fisiológicos y patológicos de los trastornos relacionados con el EPR. En este protocolo, proporcionamos un protocolo fácil de seguir para cultivar IPC primarias, que podría restaurar y mantener rápidamente las características posteriores de RP, creemos que mediante el uso de este método las personas podrían generar rápidamente células RP a partir de estudios de macistán y las pruebas de detección de medicamentos protectores que podrían facilitar el desarrollo de nuevos tratamientos para los trastornos de EPR. Una demostración paso a paso del valor hará que este método sea mucho más fácil de replicar en diferentes laboratorios que desean estudiar células RP.Para comenzar a descongelar la enzima de digestión tisular malaquad y esterilizar 1X PBS. Se complementa con 2% de penicilina y 2% de estreptomicina filtrando la solución a través de una unidad de filtro de jeringa de 0,22 micrómetros. Lave los pocillos de la placa de cultivo y los insertos de transwell con 1X PBS.Retire el PBS de los pocillos y transwell con una pipeta y agregue un mililitro de PBS fresco 1X a la cámara inferior y 600 microlitros de 10 microgramos por mililitro de solución de laminina a la cámara superior. Incubar las placas y los insertos de transpozo en esta incubadora de helicicultura durante la noche a 37 grados centígrados y 5% de dióxido de carbono. Después de eso, retire la solución laminada de la cámara superior y lave con un mililitro de PBS frío 1X dos veces antes de colocar la celda Limpie la campana de flujo laminado con 75% de etanol y luz UV.Prepare tres tubos de centrífuga estériles de 50 mililitros que contengan alrededor de 25 mililitros de etanol al 75%, tres tubos centrífugos estériles de 50 mililitros que contengan alrededor de 25 mililitros de PBS 1X y tres placas de cultivo celular estéril de 10 centímetros que contengan alrededor de 15 mililitros de PBS 1X. Remoje cuatro globos oculares porcinos en aproximadamente 15 mililitros de etanol al 75% en una placa de Petri de 10 centímetros y use un par de tijeras y fórceps para cortar todos los tejidos conectivos y músculos residuales. Para descontaminar los globos oculares, remoje y lave cuatro globos oculares en tres tubos centrífugos estériles de 50 mililitros llenos secuencialmente con etanol al 75%.Luego lave los globos oculares en tres tubos centrífugos estériles de 50 mililitros llenos con 1X PBS secuencialmente. Invierta los tubos cada minuto para lavar bien los globos oculares. Mueva los cuatro globos oculares a una placa de cultivo celular estéril de 10 centímetros que contenga 1X PBS.Recorte la superficie externa de cada globo ocular nuevamente, para eliminar el nervio óptico y los pequeños residuos. Use tijeras para hacer un pequeño corte en la intersección del limbo y la esclerótica. Y luego extirpar la córnea, el iris, el cristalino, el cuerpo vítreo y la retina neural.Transfiera el complejo de esclerótica coroidea del EPR a un nuevo plato de cultivo celular de 10 centímetros y haga cuatro cortes para aplanar la copa ocular en forma de trébol de cuatro hojas. Realice viajes en la digestión de la solución de EDTA colocando los cuatro complejos de esclerótica coroidea RPE en un nuevo plato de 10 centímetros y vertiendo 20 mililitros de solución fresca de tripsina EDTA para fusionar los complejos de esclerótica coroidea RPE. Coloque el plato en la incubadora de cultivo celular a 37 grados centígrados durante casi 30 minutos.Después de 30 minutos, saque el plato de la incubadora y agregue 20 mililitros de medios de cultivo celular precalentados con 10% FBS al plato. Para neutralizar los disparos en la solución de EDTA, utilice una pipeta de transferencia de cinco mililitros para disociar las células del EPR pipeteando suavemente varias veces. Recoja las suspensiones celulares en tubos de centrífuga de 15 mililitros y luego pipete suavemente otros 10 mililitros de medios de cultivo frescos con FBS para lavar los complejos de esclerótica de la coroides RPE en el plato.Recoger las células RPE centrifugando los tubos a 300 G durante cinco minutos a temperatura ambiente. Aspirar el SuperAgent con una pipeta y añadir otros cinco mililitros de medios de cultivo con FBS para volver a suspender las células. Centrifugar a 300 G durante otros cinco minutos.Decantar el SuperAgent y volver a suspender las células con 12 mililitros de medios de cultivo. Siguiente semilla 100, 000 a 200, 000 células por pocillo en 12 placas de cultivo de pozos o insertos de pozo. Cambie los medios de cultivo cada dos días y reduzca la concentración sérica al 1% cuando las células cubran completamente la superficie de los pocillos por inserto.Cambie los medios de cultivo cada dos días hasta que se recolecten las células. Aquí se muestran las imágenes representativas de las células primarias del EPR porcino en el día dos, el día seis y el día 10. El análisis QRTPCR de los niveles de ARNm de genes de firma en células primarias de EPR porcino, células de EPR humanas primarias, células ARPE 19 y tejido coroideo de EPR porcino se detecta mediante esta imagen gráfica.Aquí se utilizó GAP DH como el gen de limpieza para RTPCR. Se utilizaron cuatro réplicas biológicas para las células primarias del EPR porcino y un EPR 19, donde se utilizaron tres réplicas biológicas para las células del EPR humano primario y el tejido coroideo del EPR porcino. Los niveles de expresión génica en las células ARPE 19 se establecieron como controles.El análisis de sangre occidental de las proteínas RPE 65 en ARPE 19 y células RPE porcinas y en células RPE humanas primarias y tejidos coroides de RPE porcino se muestran en esta figura. Vinculin se utilizó aquí como un control de carga. Esta figura muestra un cultivo de dos semanas de células RPE porcinas en medios DMEM Basic con 1% FBS.Las células cultivadas con o sin Lamin se tiñeron con RPE 65, APTA de sodio y potasio, Z01 y Hex. Las mediciones de TR en láminas celulares, cultivadas con o sin laminina se representan en esta figura. El análisis de sangre occidental del factor de crecimiento endotelial vascular o VEGF y el factor derivado del epitelio pigmentario o PEDF en células primarias de EPR porcino y ARPE 19 se muestran en esta figura.En estas imágenes se presentan las funciones sectoriales del EPR porcino primario cultivado y las células ARPE 19. La gran parte de este protocolo es disolver las células RP de los complejos gliales de renudo del EPR. Recomendamos usar viajes frescos y solución cada vez y controlar adecuadamente el tiempo de digestión, para disolver ocho células 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.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

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 ...

Investigación

Biología

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, ...

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.

Transplante subretinal de células epiteliales del pigmento retiniano derivado de células madre embrionarias humanas en un modelo de grandes ojos de atrofia geográfica

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.

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 ...

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.

Aislamiento, cultivo e ingeniería genética de células epiteliales de pigmento primario de mamíferos para terapia génica no viral

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.

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 ...

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 ...

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.

Análisis en tiempo real de la bioenergética en células epiteliales pigmentarias de la retina humana primaria mediante respirometría de alta resolución

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.

Aislamiento de células epiteliales pigmentarias de la retina de ojos de conejillo de indias

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

Protocolo para el aislamiento de células epiteliales mamarias de tejido humano

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.

Autor destacado: Integración de la medicina oriental y occidental para el tratamiento de la mastitis granulomatosa

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.

Aislamiento y cultivo de células epiteliales mamarias humanas primarias

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