Nos gustaría dar las gracias a Andrea Portbury para la lectura crítica del manuscrito, el Laboratorio de Servicio de microscopio de UNC para obtener ayuda con el time-lapse y el NIH (subvención # R01HL061656) de apoyo financiero.

1. Prepare los granos de anticuerpos conjugados <ol><li> Un día antes de la disección, se combinan el anticuerpo de elección con los Dynabeads apropiadas (por ejemplo, rata Dynabeads IgG para un anticuerpo producido en la rata, 4 x 10 8 cuentas / ml) según las instrucciones del fabricante. Para la rata anti-CD31 anticuerpo BD (0,5 g / l, que se utiliza para aislar las células endoteliales), se añadió 3 l de anticuerpo por cada 25 l perlas, para una concentración final de 1,5 mg anticuerpo anti-CD31 y 1 x 10 7 / perlas 25 l de tampón. </li><li> Incubar los granos y de anticuerpos durante la noche a 4 ° C. </li></ol> 2. Preparación de la placa de colágeno <ol><li> Células aisladas forman túbulos más rápidamente cuando se cultivan en un gel de colágeno en comparación con simplemente una placa recubierta con colágeno. Por lo tanto, los geles de colágeno se hizo la noche antes de la disección, como se describe en 6. En el hielo en una campana de cultivo de tejidos, se combinan los siguientes reactivos (suficiente para 19 pozos): 206,4 l estéril H &lt;sub&gt; 2 S, 140,4 l de colágeno tipo I (4,08 mg / ml), 38,4 l 10x M199, y 1,15 l de NaOH 5 M. </li><li> Pipetear 20 l de este gel de colágeno en los pocillos de una placa de cultivo de tejidos de 384 pocillos. Colocar la placa en un 37 ° C incubadora de cultivo de tejido durante 30 min. </li><li> Añadir 80 l de DMEM a cada pocillo e incubar durante 15 min a 37 ° C. Retire el medio de cultivo y repetir dos veces más. </li><li> Después del enjuague final, añadir 80 l 10% de FBS / DMEM a cada pocillo e incubar durante la noche a 37 ° C. Retirar el medio de cultivo antes de la adición de las células aisladas. </li></ol> 3. Impuestos y digerir el Corazón <ol><li> La eutanasia a un ratón de disolución-embarazada en el día embrionario deseado usando una técnica de eutanasia aprobada. Todos los experimentos fueron aprobados por el Cuidado de Animales institucional y el empleo Comisión de la Universidad de Carolina del Norte en Chapel Hill. </li><li> Con el ratón en una posición supina, liberalmente rociar el lado ventral de la hembra con etanol al 70% antes de ladisección. Con unas pinzas para levantar la piel del abdomen inferior y el músculo fuera de los órganos internos, a cielo abierto la cavidad abdominal de la hembra para visualizar los órganos internos. </li><li> Sosteniendo el cuello uterino con una pinza, corta cuidadosamente caudal a los fórceps y comenzar el levantamiento de la trompa uterina de la cavidad abdominal. Mientras levanta el cuerno uterino, cortar cualquier tejido conectivo que sostiene el cuerno en su lugar. Para terminar de extirpar el cuerno uterino, corte la unión del cuello uterino con el oviducto para liberar el cuerno uterino. </li><li> Coloque el cuerno uterino en una placa de Petri que contiene frío 1x solución salina tamponada con fosfato (PBS) y enjuague según sea necesario. Cortar el cuello uterino a través de la línea media para exponer los sacos embrionarios adjuntos. </li><li> Cortar un saco embrionario en la unión de la placenta y el embrión para evitar que accidentalmente cortar el embrión y recuperar un embrión. Coloque el embrión en una segunda placa de Petri con PBS frío. Devuelva la placa de Petri con el cuerno uterino en hielo. </li><li> Decapitate el embrión para mejorar el acceso a la pared torácica. Si genotipado es necesario, cortar la cola a quitar y guardar en un tubo Eppendorf se coloca en hielo. </li><li> Con el embrión en su parte posterior, cortar y abrir la pared torácica para visualizar el corazón y los pulmones. Para evitar que se corte el corazón, hacer un corte vertical a lo largo del lado de la caja torácica, cerca de una extremidad anterior, seguido de un corte horizontal a través de la parte inferior de las costillas y, a continuación, tire suavemente de la pared del pecho hacia arriba y fuera del camino. Up hasta aproximadamente el día embrionario 13.5, la pared torácica es transparente, ayudando en la visualización. </li><li> Levante con cuidado el corazón con las pinzas y cortar los vasos a continuación. Luego, corte por encima de los grandes vasos para liberar el corazón. Si los vasos pulmonares no se cortan, los pulmones se pueden eliminar con el corazón. Por lo tanto, eliminar los tejidos extraños, como los pulmones, si es necesario. </li><li> Separar y desechar los vasos aurículas y grande de los ventrículos. Con unas tijeras, picar los ventrículos en trozos pequeños (aproximadamente 1 mm 3 &lt;/ Sup&gt;) y el lugar en aproximadamente 500 l de colagenasa (1 mg / ml en PBS estéril). Mantener las muestras en hielo hasta que los ventrículos se han picado y se coloca en la colagenasa. </li><li> Repetir los pasos anteriores hasta que los ventrículos se han extirpado de todos los embriones, la recogida de cada corazón en un tubo Eppendorf separado. Si todos los embriones tienen el mismo genotipo, corazones puedan ser agrupados en un solo tubo Eppendorf. El número de muestras procesadas en un solo tiempo puede ser limitado con base en el imán utilizado para el aislamiento, la dynamag (123-21D) que se utiliza aquí tiene un máximo de 16 tubos Eppendorf. </li><li> Coloque los ventrículos picada a 37 ° C con agitación durante 45 min. Cada 15 minutos, retire suavemente las muestras y la pipeta hacia arriba y abajo para disociar mecánicamente las células. </li></ol> 4. Aislamiento de las células endoteliales <ol><li> Pasar la solución ventrículo-colagenasa a través de un filtro de 70 micras para eliminar grupos restantes de las células. </li><li> Centrifugar las células a 400 xg durante 10 min. Descartarel sobrenadante, reemplace con aproximadamente 200 l FBS al 10% en DMEM, y la pipeta hacia arriba y abajo para disociar las células. </li><li> Repita el paso anterior una vez. Centrifugar las células a 400 xg durante 10 min después de la segunda 10% de FBS / DMEM lavado. Resuspender las células en 50 l 10% de FBS / DMEM. </li><li> Durante el paso final de la centrifugación, preparar las perlas. <ol><li> Colocar el tubo Eppendorf que contiene el anticuerpo y perlas en un imán durante 1 min. </li><li> Con el tubo en su lugar en el imán, eliminar el sobrenadante. Retire el tubo del imán y añadir aproximadamente 100 l 10% FBS / DMEM. </li><li> Colocar el tubo en el imán durante 1 min. Eliminar el sobrenadante, y se lava de nuevo con 10% de FBS / DMEM. Repita para un total de 3 lavados. </li><li> Después del último lavado, resuspender las perlas de anticuerpo-conjugados en 10% de FBS / DMEM (5 l por muestra). </li></ol></li><li> Para cada muestra de células, añadir 5 l de las perlas de anticuerpo-conjugados. Coloque las suspensiones del grano de células a 4 º C con balanceo FOr 30 min. </li><li> En una campana de flujo laminar, colocar los tubos Eppendorf que contienen las células y los granos de anticuerpos conjugados en el imán durante 1 min. Eliminar el sobrenadante, retirar los tubos del imán, y se lava con aproximadamente 100 l 10% FBS / DMEM. Repita por 5 lavados. </li><li> Después del último lavado, se colocan los tubos en el imán durante 1 min, eliminar el sobrenadante, y quitar los tubos del imán. Añadir aproximadamente 100 l de DMEM. </li><li> Colocar los tubos en el imán durante 1 min, eliminar el sobrenadante, y quitar los tubos del imán. Añadir 100-200 l precalentado 0,25% de tripsina-EDTA. Incubar las muestras a 37 ° C y CO2 al 5% durante 5 min. </li><li> Colocar los tubos en el imán durante 2 min. Transferir con cuidado el sobrenadante que contiene células a tubos Eppendorf frescos. Centrifugar durante 5 min a 400 x g. </li><li> Eliminar el sobrenadante. Resuspender las células en 80-100 l de medio de crecimiento y la placa en un 96 - o 384 pocillos placa de cultivo tratadas, dependiendo del rendimiento esperado(Aproximadamente 450-600 células de los ventrículos de embriones E14.5-E16.5). Para las células endoteliales, utilizar medio de cultivo de crecimiento endotelial (definido a continuación). </li></ol>

<ol>
	<li>Landecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Life: How Cells Became Technologies. , (2007).</li><li>Kan, C. Y., Wen, V. W., 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=Endothelial+cell+dysfunction+and+cytoskeletal+changes+associated+with+repression+of+p16(INK4a)+during+immortalization.">Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16(INK4a) during immortalization.</a> Oncogene. , (2012).</li><li>Dyer, L. A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+endothelium:+an+emphasis+on+heterogeneity.">Development of the endothelium: an emphasis on heterogeneity.</a> Semin. Thromb. Hemost. 36, 227-235 (2010).</li><li>Dong, Q. G., Bernasconi, 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+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17, 1599-1604 (1997).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3, 1085-1091 (2008).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+a+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.</a> Dev. Biol. 95, 108-114 (1983).</li><li>Arnaoutova, I., Kleinman, H. K. <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+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nat. Protoc. 5, 628-635 (2010).</li><li>Zeisberg, E. M., Potenta, S. E., Sugimoto, H., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+kidney+fibrosis+emerge+via+endothelial-to-mesenchymal+transition.">Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.</a> Journal of the American Society of Nephrology : JASN. 19, 2282-2287 (2008).</li><li>Chang, L., Noseda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+vascular+smooth+muscle+cells+from+local+precursors+during+embryonic+and+adult+arteriogenesis+requires+Notch+signaling.">Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.</a> PNAS. 109, 5993-6998 (2012).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., DeLisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, L1095-L1103 (2009).</li><li>Frauli, M., Ludwig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+a+culture+of+human+endometrial+stromal+cells+using+a+medium+containing+D-valine.">Inhibition of fibroblast proliferation in a culture of human endometrial stromal cells using a medium containing D-valine.</a> Archives of Gynecology and Obstetrics. , 241-96 (1987).</li><li>Lazzaro, V. A., Walker, R. J., 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=Inhibition+of+fibroblast+proliferation+in+L-valine+reduced+selective+media.">Inhibition of fibroblast proliferation in L-valine reduced selective media.</a> Research communications in chemical pathology and pharmacology. 75, 39-48 (1992).</li><li>Arima, S., Nishiyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

La investigación con células embrionarias primarias permite experimentos novedosos para abordar en etapas críticas del desarrollo in vitro. Sin embargo, el procedimiento de aislamiento no es trivial. Los pasos críticos para el aislamiento de cualquier tipo de células incluyen asegurar que las células están bien separados tras la digestión de colagenasa y luego de que están bien suspendidos durante los pasos de lavado. Disección mecánica mediante pipeteo en gran medida ayuda a separar las cé...

La llegada de las líneas celulares inmortalizadas ha revolucionado la biología celular básica 1. Las líneas celulares disponibles en la actualidad son derivados de una amplia gama de órganos y abarcan todos los tipos de células principales. Sin embargo, las líneas celulares establecidas tienen algunas limitaciones. El proceso de inmortalización, obviamente, cambia el comportamiento de las células, específicamente con respecto a la duración de la vida y la proliferación, sino que también puede afectar a la expresión de las proteínas inesperados, tales como las proteínas del citoesqueleto 2. Además, aunque muchos diferentes líneas celulares están disponibles, existe una diversidad significativa entre incluso un solo tipo de células dentro de un organismo entero. Las células endoteliales, en particular, muestran diversos comportamientos en función de si se alinean las arterias o las venas y el tipo de flujo está presente en el recipiente 3. Incluso más apremiante de una perspectiva de desarrollo, sin embargo, es que la gran mayoría de las líneas celulares disponibles se derivan de tejido adulto (ver, por ejemplo, tque de recogida disponible a través de ATCC). Estas líneas de células adultas probablemente no recapitulan la naturaleza dinámica de sus precursores embrionarios. El rápido cambio espacio-temporales patrones de expresión génica observados durante el desarrollo también sugieren que una célula endotelial de un órgano a la etapa de desarrollo determinado puede no comportarse de la misma manera que una célula endotelial a partir de ese mismo órgano en una etapa de desarrollo diferente. Como una alternativa al uso de líneas celulares inmortalizadas disponibles comercialmente, las células primarias pueden ser aislados a partir del tejido específico de interés. Entre las ventajas de esta técnica, estas células primarias se pueden aislar a partir de un órgano específico o incluso parte de un órgano en cualquier etapa de desarrollo específico. Además, estas células primarias se pueden aislar a partir de la amplia variedad de animales transgénicos disponibles, lo que permite el estudio in vitro del gen knockout y knock-in evitando al mismo tiempo otros problemas tales como la eficiencia de transfección en. No es sorprendente que muchostécnicas se han publicado que detalla cómo aislar determinados tipos de células 4,5. En general, estas técnicas implican la recogida de la región de interés, disociar las células de marcado, el tipo específico de célula de interés, y el aislamiento de las células para su posterior análisis. Para estudiar una población embrionario temprano de las células endoteliales coronarias, que en escala una técnica publicada previamente 4 para su uso con un órgano más pequeño. Con este procedimiento a escala reducida, podemos aislar las células endoteliales coronarias del corazón embrionario en las etapas embrionarias específicas. Estas células pueden entonces ser usados ​​en ensayos de endoteliales tradicionales, tales como los análisis de migración. Hasta que las líneas de células embrionarias tempranas cada vez más frecuentes, en colaboración con las pilas es una técnica muy valiosa.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Timed-pregnant mice</td>
			<td></td>
			<td></td>
			<td>To be dissected at the embryonic stage of interest</td>
		 </tr>
		 <tr>
			<td>Anti-mouse CD31</td>
			<td>BD Bioscience</td>
			<td>553370</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat IgG Dynabeads</td>
			<td>Invitrogen</td>
			<td>110-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagen</td>
			<td>BD Bioscience</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Cellgro </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich </td>
			<td>F2442 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endothelial cell growth medium</td>
			<td>(made in lab as described in notes)</td>
			<td></td>
			<td>DMEM containing 20% FBS, 5 &mu;M &beta;-mercaptoethanol, 50 &mu;g/ml ECG, and 1x penicillin/streptomycin</td>
		 </tr>
		 <tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ECGS</td>
			<td>Biomedical Technologies, Inc.</td>
			<td>BT-203</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>384-well culture plate</td>
			<td>Greiner</td>
			<td>T-3037-6</td>
			<td>Plate was prepared with a thin collagen gel, as described in 6; working surface area 10 mm2</td>
		 </tr>
		 <tr>
			<td>Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>Use at a final concentration of 1.5 mg/ml</td>
		 </tr>
		 <tr>
			<td>M199, 10x</td>
			<td>Invitrogen</td>
			<td>11825-015</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Syto-16</td>
			<td>Invitrogen </td>
			<td>S7578 </td>
			<td>Used as directed in 13 </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Stereoscopic microscope</td>
			<td>Nikon </td>
			<td>SMZ645</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell culture incubator</td>
			<td>Thermo </td>
			<td>3110 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>DynaMag</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Table-top centrifuge</td>
			<td>Thermo</td>
			<td>75002430</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation of embryonic ventricular endothelial cells

Cultivo celular ha mejorado en gran medida nuestra capacidad para evaluar las poblaciones individuales de las células bajo condiciones de cultivo innumerables. Mientras que las líneas celulares inmortalizadas ofrecen ventajas significativas para su facilidad de uso, estas líneas celulares no están disponibles para todos los tipos de células potenciales. Al aislar las células primarias de una región específica de interés, en particular de un ratón transgénico, se pueden realizar estudios más matizadas. La técnica básica consiste en la disección del órgano u órgano parcial de interés (por ejemplo, el corazón o de una región específica del corazón) y disociar el órgano a las células individuales. Estas células se incuban a continuación con perlas magnéticas conjugadas con un anticuerpo que reconoce el tipo de célula de interés. Las células de interés se pueden aislar con el uso de un imán, con una breve incubación con tripsina disociar las células de las perlas. Estas células aisladas pueden cultivarse a continuación, y se analizaron como se desee. Esta técnica fue diseñada originalmente para órgano ratón adultos pero se puede escalar fácilmente hacia abajo para su uso con órganos embrionarios, como se demuestra en este documento. Debido a que nuestro interés se centra en el desarrollo de la vasculatura coronaria, queríamos estudiar esta población de células durante las fases embrionarias específicas. Por lo tanto, el protocolo original tuvo que ser modificado para ser compatible con el pequeño tamaño de los ventrículos embrionarias y el bajo rendimiento potencial de las células endoteliales en estas etapas de desarrollo. Utilizando este enfoque a escala reducida, hemos evaluado la remodelación del plexo coronaria en las células endoteliales ventriculares embrionarias transgénicas.

Uso de un anticuerpo endotelial-específico que reconoce CD31, las células endoteliales se aislaron de los ventrículos del embrionarias (E) 13,5 (Figura 1A) y 18.5 (Figuras 1B-1D) embriones. Cuando se cultiva en una placa de cultivo sin tratar, estas células siguen siendo redondeadas y formarían un patrón de adoquines si confluente cerca (Figura 1A). Diluir colágeno también se ha utilizado para recubrir los pozos, y mientras que las células endoteliales se adher...

Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

Isolation of Embryonic Ventricular Endothelial Cells

Cultivo celular primaria es una técnica útil para el análisis de poblaciones específicas de células, en particular a partir de embriones de ratones transgénicos en etapas de desarrollo específicas. En esto, los ventrículos embrionarias se disecan y se disociaron, y los granos de anticuerpos conjugados reconocen y se separan las células endoteliales para su posterior análisis.

Aislamiento de células endoteliales embrionarias ventriculares

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

isolation-of-embryonic-ventricular-endothelial-cells

Research

JoVE Journal

Biology

9.3K vistas.  University of North Carolina at Chapel Hill. Cultivo celular primaria es una técnica útil para el análisis de poblaciones específicas de células, en particular a partir de embriones de ratones transgénicos en etapas de desarrollo específicas. En esto, los ventrículos embrionarias se disecan y se disociaron, y los granos de anticuerpos conjugados reconocen y se separan las células endoteliales para su posterior análisis.

Video: Isolation of Embryonic Ventricular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

El aislamiento de las células endoteliales de vena umbilical (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Investigación

Biología

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Células madre embrionarias-las células endoteliales derivadas de tratamiento de la isquemia Miembro Posterior

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Aislamiento de células endoteliales valvular

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Aislamiento de las células murinas de la válvula endoteliales

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Aislamiento de células endoteliales de ratón coronaria

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...