Esta investigación es apoyada por el premio CARRERA NSF, la Fundación Hartwell, y la Asociación Americana del Corazón (# 0830384N).

1. Preparación <ol><li> Autoclave en un instrumento bandeja cubierta los siguientes elementos: <ol><li> Dentada con fórceps del tejido - Para el manejo de los tejidos folleto </li><li> Tijeras de tejidos (8 cm) - Para cortar el tejido folleto y las cúspides </li><li> Los hisopos de algodón - Para aislar la capa endotelial de la hoja o cúspide </li></ol></li><li> Hacer una solución estéril de colagenasa <ol><li> Añadir 4,0 gramos de polvo DMEM a 250 ml de 18 mW de agua. </li><li> Añadir 1,11 gramos de bicarbonato de sodio. </li><li> Añadir (600 U / mL) 180.000 unidades de la colagenasa. </li><li> Añadir al 1% (3 ml) de penicilina / estreptomicina. </li><li> Ajustar el pH de la solución a 7,2. </li><li> Se lleva la solución hasta 270 ml. </li><li> Esterilizar la solución al pasar por un filtro de 0,2 micras. </li><li> Añadir un 10% (30 ml) de suero fetal bovino estéril. </li></ol></li><li> Haz medio estéril de las células endoteliales <ol><li> Añadir 6,7 gramos de polvo DMEM a 400 ml de 18 mW de agua. </li><li> Añadir 1,85 gramos de bicarbonato de sodio. </li><li> Añadir (50 U / mL) 25.000 unidades de heparina. </li><li> Agregue 1% (5 ml) de penicilina / estreptomicina. </li><li> Ajustar el pH de la solución a 7,2. </li><li> Se lleva la solución hasta 450 ml. </li><li> Esterilizar la solución al pasar por un filtro de 0,2 micras. </li><li> Añadir un 10% (50 ml) de suero fetal bovino estéril. </li></ol></li><li> Haz medio estéril de células intersticiales <ol><li> Añadir 13,37 gramos de polvo DMEM a 800 ml de 18 mW de agua. </li><li> Añadir 3,7 gramos de bicarbonato de sodio. </li><li> Agregue 1% (10 ml) de penicilina / estreptomicina. </li><li> Ajustar el pH de la solución a 7,2. </li><li> Se lleva la solución hasta 900 ml. </li><li> Esterilizar la solución al pasar por un filtro de 0,2 micras. </li><li> Añadir un 10% (100 ml) de suero bovino estéril. </li></ol></li></ol> 2. El aislamiento de valvas de las válvulas del corazón <ol><li> Impuestos Especiales de la raíz aórtica inmediatamente y aséptica del corazón después del sacrificio. </li><li> Enjuague bien la raíz aórtica de sangre fría con DPBS estéril. Es imperativo eliminar todos los componentes de la sangre tan pronto como sea posible para limitar la muerte VEC y la contaminación bacteriana. Antibióticos y antimicóticos no son aconsejables en esta etapa, ya que son potencialmente dañinas para el VEC. </li><li> Aislar valvas de las válvulas (3 por válvula) directamente de la raíz y el lugar en un tubo cónico de 15 ml estéril llena con 12 ml de DPBS frío. Agite varias veces para eliminar los residuos y relleno con DPBS fresco. </li><li> Transporte al laboratorio en hielo. </li><li> A su llegada al laboratorio, colocar el recipiente con el tejido debajo de la campana estéril. </li></ol> 3. El aislamiento de la capa endotelial <ol><li> Llenar un plato de 35 mm estéril con 3 ml de solución de colagenasa frío por la válvula (3 hojas). </li><li> Coloque los tres folletos del tubo de 15 ml en el plato lleno con la solución de colagenasa. </li><li> Incubar el tejido durante 5-10 minutos a 37 ° C. </li><li> Retire con cuidado la capa endotelial de rotación de un hisopo estéril seco sobre la superficie de la hoja. El sentido de giro y la cantidad de cizalladura aplicada es fundamental para la pureza de la muestra. La rotación del algodón debe estar en una dirección opuesta al movimiento lineal de la mano la creación de un corte controlado. Este corte es lo que levanta las células endoteliales del tejido. La cantidad de fuerza aplicada debe ser suficiente para sentir la resistencia del tejido, pero no penetran en la membrana basal. </li><li> De vez en cuando, aplique el hisopo dentro de la solución de colagenasa para separar las células de las fibras de la punta. Después de hisopado se haya completado, la textura de la capa endotelial debe sentirse un poco más suave que antes. </li><li> Recoger la suspensión de células / colagenasa y la transferencia a un nuevo tubo de 15 ml estéril. </li><li> Centrifugar los tubos a 1000 rpm durante 5 minutos para que sedimenten las células aisladas y aspirar el sobrenadante. Si el aislamiento de células intersticiales, así, lleve a cabo ese protocolo, mientras que estas células se centrifugan. </li><li> Añadir 3 ml de medio porcino endotelial a la tubos de 15 ml, centrifugar por segunda vez, y los medios de comunicación aspirado. Esta segunda centrifugación ayuda a filtrar algunos de los materiales no deseados, como las fibras de la punta. </li><li> Vuelva a suspender las células endoteliales se centrifuga en 5 ml de medio porcino endoteliales y las células de la placa en un pre-recubiertos frasco T-25 con colágeno (use un frasco por cada tubo de centrífuga). </li><li> Deje que las células crecen por lo menos 2-3 días antes de cambiar el medio endoteliales. Esto ayuda a que las células se dividen y se recuperan desde el proceso de aislamiento es muy duro y el rendimiento celular puede ser baja. Es muy importante para las células paso cerca de la confluencia ya que la inhibición de contacto podría conducir a la transformación celular. </li></ol> 4. Preparación de la placa de cultivo de 60 mm de aislamiento secundarios específicos <ol><li> La línea de 60 mm de vidrio placas de Petri con papel de aluminio (2 platos de vidrio por leaflet). El papel de aluminio ayuda a eliminar la parafina, para que el plato Petri de vidrio se puede reutilizar para otros aislamientos. </li><li> Lugar bolas de parafina en los platos, hasta la mitad, y la cobertura de tratamiento en autoclave. </li><li> Una vez que el autoclave se ha completado y la parafina se funde, se mueve el conjunto de plato para una superficie plana y fría. A medida que la parafina se enfría, se endurece y crea una capa que apoyará a los pinchazos de la aguja. </li><li> Después de 30 minutos, el plato esterilizada se puede utilizar como una cámara de aislamiento para inmovilizar el tejido folleto. </li></ol> 5. El aislamiento de lado de la capa endotelial específico <ol><li> Retire los folletos de los tubos de 15 ml y coloque en un plato de cultivo preparados para el aislamiento de 60 mm secundarios específicos. </li><li> Manipular el volante para que el lado ventricularis está boca abajo sobre la superficie de la parafina, dejando la parte fibrosa expuestos. Pin de los bordes de la hoja para exponer la capa endotelial. </li><li> Ponga unas gotas de la colagenasa en frío en cada superficie (arriba de cara) endotelial e incubar el tejido durante 5-10 minutos a 37 ° C. </li><li> Al igual que antes, retire suavemente la capa endotelial de rotación de un hisopo estéril seco sobre la superficie de la hoja. El sentido de giro y la cantidad de cizalladura aplicada es fundamental para la pureza de la muestra. La rotación del algodón debe estar en una dirección opuesta al movimiento lineal de la mano la creación de un corte controlado. Este corte es lo que levanta las células endoteliales de los tejidos y nada más. La cantidad de fuerza aplicada debe ser suficiente para sentir la resistencia del tejido, pero no penetra en el tejido. </li><li> De vez en cuando, aplique el hisopo dentro de la solución de colagenasa para separar las células de las fibras de la punta. Después de hisopado se haya completado, la textura de la capa endotelial debe sentirse un poco más suave que antes. </li><li> Recoger la suspensión de células / colagenasa y la transferencia a un nuevo tubo de 15 ml estéril. Indican especificidad lado de la etiqueta (folletos de la misma válvula se pueden agrupar entre sí). </li><li> La transferencia de los prospectos para una placa de cultivo nuevo para que el lado ventricularis quedará al descubierto y repita los pasos (5.3 a 5.6). </li><li> Una vez que todos suspensión celular / colagenasa se recoge, centrifugue los tubos a 1000 rpm durante 5 minutos para que sedimenten las células aisladas y aspirar el sobrenadante. Si el aislamiento de células intersticiales, así, lleve a cabo ese protocolo, mientras que estas células se centrifugan. </li><li> Añadir 3 ml de medio porcino endotelial a los tubos de centrífuga por segunda vez, y aspirar los medios de comunicación. Esta segunda centrifugación ayuda a filtrar algunos de los materiales no deseados, como las fibras de la punta. </li><li> Vuelva a suspender las células endoteliales se centrifuga en 5 ml de medio porcino endoteliales y las células de la placa en un pre-recubrimiento T-25 matraz con collage (use un frasco por cada tubo de centrífuga). </li><li> Deje que las células crecen por lo menos 2-3 días antes de cambiar el medio endoteliales. Esto ayuda a que las células se dividen y se recuperan desde el proceso de aislamiento es bastante dura. Si nota que el rendimiento sea muy bajo, considere a un matraz pequeño o una placa y desde las células de adhesión celular promueve el crecimiento celular. Recuerde que paso cerca de la confluencia ya que la inhibición de contacto podría conducir a la transformación celular. </li></ol> 6. El aislamiento de las células intersticiales <ol><li> Llene un tubo de centrífuga estéril 15 ml con 10 ml de solución de colagenasa por válvula (3 folletos). </li><li> Después de limpiando los folletos de las células endoteliales, inmediatamente el lugar en el tubo de 15 ml con la adecuada solución de colagenasa. </li><li> Se incuba durante aproximadamente 12 a 18 horas (agitar suavemente, si lo desea). </li><li> Mezclar el tejido degradado suavemente con una pipeta serológica hasta que la suspensión de células / colagenasa se homogeniza. Esta homogeneización ayuda a romper el tejido y la liberación de las células intersticiales. </li><li> Centrifugar el tejido digerido durante 5 minutos a 1000 rpm y aspirar el sobrenadante. </li><li> Añadir 5 ml de medio porcino intersticial a los tubos de 15 ml, centrifugar por segunda vez, y aspirar sobrenadante. </li><li> Vuelva a suspender las células endoteliales se centrifuga en 5 ml de medio porcino intersticial y la placa de las células en un frasco T-75 (uso de un frasco por cada tubo de centrífuga). </li><li> Deje que las células crecen por lo menos 1-2 días antes de cambiar el medio de células intersticiales. Habrá restos mucho más tejido que con las células endoteliales, pero que se espera. Las células también debe crecer hasta la confluencia más rápido que las células endoteliales, debido a la producción de células y su naturaleza. </li></ol> 7. Imágenes representativas <img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Figura 1" /> Figura 1. La morfología de las células aisladas de 2-3 días después de la aislamiento. (A) VEC presentan una morfología típica endoteliales y formar grupos para promover el crecimiento. (B) VIC morfología es similar a miofibroblastos que por lo general en forma de huso y dispersándose por todo el frasco. <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Figura 2" /> Figura 2. La morfología de las células aisladas en la confluencia. (A) VEC presentan una morfología típica endoteliales, que son generalmente de adoquines y el contacto inhibe el crecimiento. (B) VIC morfología es similar a miofibroblastos que por lo general en forma de huso y no inhibe el crecimiento de contacto. <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Figura 3" /> Figura 3. Las características de la función de las células aisladas 6. (A) VEC están asociados con altos niveles de captación de LDL acetilada (rojo). (B) VIC están asociados con bajos niveles de captación de LDL acetilada. <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Figura 4" /> Figura 4. Marcadores celulares de células aisladas 6. (A) fenotipo VEC se contribuye a una tinción positiva para el factor von Willebrand (verde), azul (núcleos). (B) fenotipo VIC es una contribución a la tinción negativa de factor von Willebrand. <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Figura 5" /> Figura 5. Marcadores celulares de células aisladas. (A) fenotipo VEC es una contribución a la tinción negativa para el alfa SMA (verde), azul (núcleos). (B) fenotipo VIC se contribuye a una tinción positiva para el SMA alfa. <table border="1"><tbody><tr><td> Agente de la disociación </td><td> La disociación Técnica </td><td> La recogida de células </td><td> Cantidad de células </td><td> Célula Pureza </td><td> Contaminación </td></tr><tr><td> EDTA (3 mM) sin CaCl2 </td><td> 5, 20, 60 min. Además antes de CaCl2 </td><td> 20, 60, 120 min. antes de la recolección </td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td> EDTA (6 mm) sin CaCl2 </td><td> 5, 20, 60 min. Además antes de CaCl2 </td><td> 20, 60, 120 min. antes de la recolección </td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td> Tripsina-EDTA (0,5 g / L) </td><td> 5, 10, 15 min. antes de la desactivación </td><td> Medio de recogida inmediatamente </td><td> + </td><td> + </td><td> + + </td></tr><tr><td> Colagenasa II (300 U / mL) </td><td> 5, 10, 15 min. antes de la desactivación </td><td> Medio de recogida inmediatamente </td><td> -, +, + + </td><td> -, + +, + </td><td> + </td></tr><tr><td> Colagenasa II (600 U / mL) </td><td> 5, 10, 15 min. antes de la desactivación </td><td> Medio de recogida inmediatamente </td><td> +, + +, + + + </td><td> + +, + +, - </td><td> - </td></tr></tbody></table> Tabla 1. Resultados preliminares de los protocolos de las células endoteliales valvular aislada.

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

La comprensión de la biología valvular se ha visto afectada por las dificultades técnicas de aislamiento y cultivo de poblaciones puras de células endoteliales valvular. Técnicas típicas de aislamiento implican digestión enzimática de la matriz subyacente basal o químicos de disociación de las uniones adhesivas endotelial 2,3. Los experimentos preliminares de aislamiento se evaluaron cualitativamente mediante la variación de los agentes de la disociación y el período de incubación. Los resultado...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
<tr>
<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
<tr>
<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
<tr>
<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
<tr>
<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
<tr>
<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25384-092</td>
<td>&nbsp;</td>
<tr>
<td>60x15 Glass Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-310</td>
<td>&nbsp;</td>
<tr>
<td>Paraffin Embedding Wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
<td>&nbsp;</td>
<tr>
<td>Precision Glide Needles</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>305165</td>
<td>&nbsp;</td>
<tr>
<td>500 mL Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-985</td>
<td>&nbsp;</td>
<tr>
<td>1L Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-986</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11023-15</td>
<td>&nbsp;</td>
<tr>
<td>FSC Tweezers #5</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of valvular endothelial cells

Las válvulas del corazón es el único responsable de mantener el flujo de sangre unidireccional a través del sistema cardiovascular. Estos tejidos finos y fibrosos están sometidas a importantes tensiones mecánicas que pueden abrir y cerrar miles de millones de veces durante un período de vida. La increíble resistencia de estos tejidos se debe a la residencia del endotelio valvular (VEC) y las células intersticiales (VIC) que constantemente la reparación y remodelación en respuesta a las señales mecánicas y biológicas. Sólo recientemente hemos comenzado a comprender los comportamientos únicos de estas células, por lo que la experimentación in vitro ha jugado un papel clave. Especialmente difícil es el aislamiento y cultivo de VEC. Especial cuidado se debe utilizar desde el momento en que se extirpa el tejido del huésped a través de revestimiento final. Aquí se presentan los protocolos para el aislamiento directo, aislamiento secundarios específicos, la cultura, y la verificación de las poblaciones puras de VEC. Usamos la digestión enzimática seguida de una técnica de raspado suave esponja para separar las células en la superficie. Estas células son luego recogidos en un tubo y se centrifuga en una pastilla. El pellet se resuspende entonces y se colocaron en frascos de cultivo pre-recubiertos con colágeno I de la matriz. Fenotipo VEC es confirmado por el contacto inhibe el crecimiento y la expresión de marcadores endoteliales específicos, tales como PECAM1 (CD31), Factor Von Willebrand (FVW), y la expresión negativa de la actina de músculo liso alfa (α-SMA). Las características funcionales de VEC se asocian con altos niveles de LDL acetilada. A diferencia de las células endoteliales vasculares, VEC tiene la capacidad única de transformarse en mesénquima, que normalmente ocurre durante la formación embrionaria de la válvula 1. Esto también puede ocurrir durante el confluente de manera significativa después de prolongados en cultivo in vitro, así que el cuidado debe ser de paso o cerca de su confluencia. Tras el aislamiento VEC, poblaciones puras de VIC pueden ser fácilmente adquiridas.

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

Isolation of Valvular Endothelial Cells

Ofrecemos un método para aislar y cultivar las poblaciones puras de células endoteliales de la válvula del corazón (VEC). VEC puede ser aislada de uno y otro lado de la cúspide o el prospecto e inmediatamente después, las células intersticiales de base (CIV) aislada es muy sencillo.

Aislamiento de células endoteliales valvular

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

Research

JoVE Journal

Biology

20.3K vistas.  Cornell University. Ofrecemos un método para aislar y cultivar las poblaciones puras de células endoteliales de la válvula del corazón (VEC). VEC puede ser aislada de uno y otro lado de la cúspide o el prospecto e inmediatamente después, las células intersticiales de base (CIV) aislada es muy sencillo.

Video: Isolation of Valvular 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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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 Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Aislamiento de microvascular endotelial Tubos de arterias de resistencia Ratón

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

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 and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Aislamiento y cultivo de células endoteliales aórticas primarias de cerdos en miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...