The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Ética declaración: Los vasos sanguíneos utilizados en este estudio fueron cosechadas como material excedente obtenido a partir de cadáveres caninos frescas (n = 4) de los perros sanos sacrificados para otras investigaciones no relacionado (política de la Universidad 3R). vasos sanguíneos anormales (derivación portosistémica intra y extrahepáticas, n = 1 cada uno) fueron cosechadas después de la muerte tras el consentimiento informado de los propietarios de los perros presentados a la Clínica Universitaria de Animales de Compañía de la Universidad de Utrecht. 1. Aislamiento y cultivo de células endoteliales primaria canina <ol><li> Pre-coat placas de 6 pocillos con 2 ml / pocillo 0,5% w / v de gelatina y se deja durante 2 horas en una incubadora con una atmósfera humidificada de 5% de CO2 a 37 ° C. Eliminar el exceso de solución de gelatina antes de la siembra de las células primarias. </li><li> Asépticamente eliminar el vaso sanguíneo (s) de interés (por ejemplo, aorta, vena cava, la vena porta) de un fresco canino cadáver (Figura 1A). Mantener la anatomía del sistema de vasosmediante la colocación de pinzas rectas en ambos extremos del vaso de interés. Evitar la manipulación innecesaria de tejido con unas pinzas para evitar daños en las células endoteliales. <ol><li> Cortar el recipiente sujetado en ambos extremos con tijeras quirúrgicas y retirar del cadáver. Transporte en solución salina equilibrada de Hank (HBSS) en hielo. NOTA: A la eslora del buque de aproximadamente 5 cm es el preferido para este procedimiento, pero los que miden 1 cm también proporcionará suficientes células para la cultura. </li></ol></li><li> Transferir el vaso sanguíneo a una placa de Petri llena de HBSS enfriado con hielo. Usando tijeras quirúrgicas, eliminar cualquier tejido adherente y la grasa de la parte exterior del recipiente, manteniendo el recipiente en sí intacta (Figura 1B). Asegúrese de que el buque es claramente visible en todo momento cuando se corta: tirar de un lado el tejido circundante con una pinza o una pinza para la visión óptima. <ol><li> Cierre todas las ramas de la embarcación con ligaduras (por ejemplo, poliglactina 3-0) y posteriormente eliminarloscon unas tijeras quirúrgicas o un bisturí. </li></ol></li><li> Introducir cuidadosamente un extremo recipiente con un fórceps curvos mosquito Halsted, sujete la punta de las pinzas en el otro extremo del recipiente y luego retraer, invirtiendo lentamente el recipiente hasta que esté completamente dentro hacia fuera (Figura 1C-G). El exterior ahora consiste en la capa de células endoteliales. Si cualquier dificultad se encuentra en la inversión del recipiente, sumergir en HBSS de nuevo para reducir la fricción. </li><li> Coloque suturas en bolsa de tabaco en ambos extremos de la vasija para cerrarla completamente y para evitar la exposición de cualquier tejido vascular no endotelial para el procedimiento de digestión (Figura 1H). Usa los extremos de ligadura para manipular el recipiente invertido. </li><li> Si la digestión y la cultura se lleva a cabo más tarde, criopreservar el recipiente invertido antes de la digestión de protocolo (paso 1.7). <ol><li> Coloque el recipiente invertido en un criovial y se llenan de cultivo celular medio de congelación. Congelar hasta -80 ° C utilizando un co congelaciónntainer. Almacenar a -80 ° C, si los barcos se van a utilizar dentro de una semana. Para el almacenamiento a largo plazo, en lugar crioviales -180 ° C. Al realizar el aislamiento CE, descongelar los crioviales rápidamente en un baño de agua (37 ° C) y colocar inmediatamente en HBSS enfriado con hielo. Proceder como se indica en 1.7. NOTA: Tenga en cuenta que el rendimiento total de los CE después del aislamiento será más bajo debido a la pérdida de viabilidad durante el proceso de congelación. </li></ol></li><li> Transferir el buque a un tubo de 50 ml y enjuague dos veces en HBSS para eliminar los eritrocitos (o medio de congelación residual en caso de descongelación) (Figura 1I). </li><li> Digerir el recipiente en un tubo de 50 ml con una solución de 30 ml de colagenasa tipo II (0,15 U / ml) y dispasa (0,15 U / ml) en células caninas medio de crecimiento endotelial (CECGM) durante 1 hora a 37 ° C con suave intermitente agitación. </li><li> Retire el recipiente del tubo y centrifugar la suspensión celular durante 5 minutos a 250 x g. </li><li> Resuspender el sedimento celular en un CECGMnd semilla de 2 ml de suspensión celular por pocillo (1 - 3 pozos dependiendo del tamaño del recipiente). Cultivar las células a 37 ° C en una atmósfera húmeda con 5% de CO2 en aire y el cambio del medio de cultivo dos veces por semana. </li><li> El paso de las células cuando una confluencia del 70 - 80% se alcanza. <ol><li> Lavar las células con HBSS precalentado para eliminar las células muertas o no inscritos. Añadir 200 enzima de disociación celular recombinante l (1x) a cada pocillo y colocar de nuevo en la incubadora durante 5 min o hasta que se separan todas las células. </li><li> Transferir las células a un tubo de 15 ml y dejar de tripsinización añadiendo 10 ml de medios de cultivo (CEGM) incluyendo 10% suero fetal de ternera (FCS). Centrifugar durante 5 min a 250 x g. Continuar la cultura en una placa de gelatina pre-recubiertos o frasco. </li></ol></li></ol> 2. Caracterización <ol><li> Cultura células endoteliales aórticas canina (CnAOECs) y comparar la morfología de las líneas de células primarias y comerciales dos veces por semana con un microscopio. NOTA: Tlo típico patrón de crecimiento en los EC parches mejor se puede observar cuando se alcanza una confluencia de&gt; 70%. <ol><li> Cultura CnAOECs en CECGM en un matraz T75 0,5% w / v de gelatina pre-revestido. células de paso una vez a la semana cuando la confluencia puede alcanzar el 70 - 80%. <ol><li> Para los pases, se lavan las células una vez con HBSS precalentado y añadir 1 ml de la enzima de disociación celular recombinante (1x). Colocar el matraz de nuevo en la incubadora durante 5 min o hasta que se separan todas las células. Inactivar la tripsina mediante la adición de medio de cultivo 10 ml (CEGM) que incluye 10% de FCS. Centrifugar la suspensión celular durante 5 min a 250 xg, descartar el sobrenadante y resuspender las células en medio de cultivo 1 ml. </li><li> Tomar una parte alícuota de 10 l de la suspensión de células y diluir 1: 1 con 0,4% de azul de tripano. Contar las células usando un contador de células automatizado y se resiembra 4,0 x 10 5 células viables en un nuevo matraz T75. Añadir 10 ml de CECGM pre-calentado al matraz y la cultura a 37 ° C en una atmósfera húmeda con 5% de CO2. </li></ol></li></ol></li><li> Aislar el ARN del paso 1 de los CaPECs cultivadas y CnAOECs. <ol><li> Recoge al menos 1 x 10 3 células como un gránulo, añadir 20 l de reactivo de preparación de la muestra (SPR) y se incuba durante 1 min para lisar las células. Después de la incubación, recoger cuidadosamente el lisado celular que contiene el ARN total y se almacena a -70 ° C. </li></ol></li><li> Convertir ARNm en ADNc usando un kit de síntesis de ADNc (por ejemplo, iScript) siguiendo las instrucciones del fabricante. ADNc se almacena a 4 ° C durante hasta una semana, oa -20 ° C a largo plazo hasta que esté listo para realizar la qPCR. </li><li> Medir la expresión génica de la CD31 marcador endotelial para confirmar el origen de las células endoteliales. Realice la configuración experimental y cuantificación utilizando las directrices de actas MIQE 10. Para la normalización, medir la expresión de genes de referencia GAPDH, rps19, y B2MG 11. <ol><li> Preparar una dilución de 10 veces del cDNA obtenido en agua libre de nucleasa. </li><li> Prepararuna dilución de 4 veces a partir de muestras de cDNA mancomunados a la línea estándar y el uso de agua libre de nucleasa como un control sin plantilla. Diluir las muestras cinco veces para llegar a un total de dilución de 50 veces para las reacciones de qPCR. Pipeta de 10 l reacciones por duplicado en un formato de 384 pocillos usando 4 l de cDNA y 6 l del fluoróforo se mezcla con 20 pmol de cebador directo y reverso (Tabla 1). </li><li> Configurar el programa para 95 ° C durante 5 min para la activación de la polimerasa Taq, seguido por 39 ciclos de 10 segundos a 95 ° C para la desnaturalización, y 30 seg a Tm para el recocido y elongación. La Tm para cada conjunto de cebadores se muestra en la Tabla 1. </li><li> Ejecutar un análisis de curva de fusión después de cada carrera que exista un sólo producto se amplifica. Normalizar los niveles de expresión de las muestras usando la cantidad relativa media de los genes de referencia, y calcular el Ct si la eficiencia de reacción está entre 95% y 105%. </li></ol></li><li> Evaluar la funcionalidad CE con un angiogenesis ensayo. <ol><li> Añadir 10 l de matriz extracelular a cada pocillo de un portaobjetos de angiogénesis pre-enfriado y extender con una punta de pipeta para cubrir la superficie del pozo. Mantener la matriz extracelular en hielo y evitar la introducción de burbujas de aire en el gel. Solidificar el gel mediante la colocación de la corredera en una placa de Petri con agua empapada toallas de papel en una incubadora a 37 ° C con 5% de CO2 durante 30 min. </li><li> Añadir 1,0 x 10 4 primaria EC en 50 l de Crecimiento Endotelial de medio por pocillo. Incubar el portaobjetos durante 6 horas a 37 ° C con 5% de CO 2. </li><li> Después de 6 horas tomar fotografías con una ampliación de 20X, asegurándose de incluir todo el bien en la imagen. </li></ol></li></ol>

<ol>
	<li>Haidara, M. A., Assiri, A. S., Yassin, H. Z., Ammar, H. I., Obradovic, M. M., Isenovic, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Failure+Models:+Traditional+and+Novel+Therapy.">Heart Failure Models: Traditional and Novel Therapy.</a> Curr. Vasc. Pharmacol. 13 (5), 658-669 (2015).</li><li>van Steenbeek, F. G., van den Bossche, L., Leegwater, P. A., Rothuizen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+liver+shunts+in+dogs+elucidate+pathways+regulating+embryonic+development+and+clinical+disorders+of+the+portal+vein.">Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein.</a> Mamm. Genome. 23 (1-2), 76-84 (2012).</li><li>Murai, A., Asa, S. A., Kodama, A., Hirata, A., Yanai, T., Sakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+phosphorylation+of+the+mTORC2/Akt/4E-BP1+pathway+in+newly+derived+canine+hemangiosarcoma+cell+lines.">Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines.</a> BMC Vet. Res. 8 (1), 128 (2012).</li><li>Boilson, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+circulating+progenitor+cells+in+left+ventricular+dysfunction.">Regulation of circulating progenitor cells in left ventricular dysfunction.</a> Circ. Heart Fail. 3 (5), 635-642 (2010).</li><li>Gonzalez-Miguel, J., Morchon, R., Siles-Lucas, M., Simon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolysis+and+proliferative+endarteritis:+two+related+processes+in+chronic+infections?+The+model+of+the+blood-borne+pathogen+Dirofilaria+immitis.">Fibrinolysis and proliferative endarteritis: two related processes in chronic infections? The model of the blood-borne pathogen Dirofilaria immitis.</a> PLoS One. 10 (4), e0124445 (2015).</li><li>Sacks, T., Moldow, C. F., Craddock, P. R., Bowers, T. K., Jacob, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+radicals+mediate+endothelial+cell+damage+by+complement-stimulated+granulocytes.+An+in+vitro+model+of+immune+vascular+damage.">Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. An in vitro model of immune vascular damage.</a> J. Clin. Invest. 61 (5), 1161-1167 (1978).</li><li>Aranguren, X. L., 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=Unraveling+a+novel+transcription+factor+code+determining+the+human+arterial-specific+endothelial+cell+signature.">Unraveling a novel transcription factor code determining the human arterial-specific endothelial cell signature.</a> Blood. 122 (24), 3982-3992 (2013).</li><li>van Balkom, B. 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+cells+require+miR-214+to+secrete+exosomes+that+suppress+senescence+and+induce+angiogenesis+in+human+and+mouse+endothelial+cells.">Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.</a> Blood. 121 (19), 3997-4006 (2013).</li><li>Crampton, S. P., Davis, J., Hughes, C. C. <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+human+umbilical+vein+endothelial+cells+(HUVEC).">Isolation of human umbilical vein endothelial cells (HUVEC).</a> J. Vis. Exp. (3), e183 (2007).</li><li>Bustin, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIQE+precis:+Practical+implementation+of+minimum+standard+guidelines+for+fluorescence-based+quantitative+real-time+PCR+experiments.">MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.</a> BMC Mol. Biol. 11, 74 (2010).</li><li>Brinkhof, B., Spee, B., Rothuizen, J., Penning, L. 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+and+evaluation+of+canine+reference+genes+for+accurate+quantification+of+gene+expression.">Development and evaluation of canine reference genes for accurate quantification of gene expression.</a> Anal. Biochem. 356 (1), 36-43 (2006).</li><li>Heishima, 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=MicroRNA-214+and+MicroRNA-126+Are+Potential+Biomarkers+for+Malignant+Endothelial+Proliferative+Diseases.">MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases.</a> Int. J. Mol. Sci. 16 (10), 25377-25391 (2015).</li><li>Liu, M. M., Flanagan, T. C., Lu, C. C., French, A. T., Argyle, D. J., Corcoran, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+characterisation+of+canine+mitral+valve+interstitial+and+endothelial+cells.">Culture and characterisation of canine mitral valve interstitial and endothelial cells.</a> Vet. J. 204 (1), 32-39 (2015).</li><li>van den Bossche, L., van Steenbeek, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+congenital+portosystemic+shunts:+disconnections+dissected.">Canine congenital portosystemic shunts: disconnections dissected.</a> The Veterinary Journal. 211, 14-20 (2015).</li><li>Sobczynska-Rak, A., Polkowska, I., Silmanowicz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+Vascular+Endothelial+Growth+Factor+(VEGF)+levels+in+the+blood+serum+of+dogs+with+malignant+neoplasms+of+the+oral+cavity.">Elevated Vascular Endothelial Growth Factor (VEGF) levels in the blood serum of dogs with malignant neoplasms of the oral cavity.</a> Acta Vet. Hung. 62 (3), 362-371 (2014).</li><li>Zhang, Q., 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=In+vitro+and+in+vivo+study+of+hydralazine,+a+potential+anti-angiogenic+agent.">In vitro and in vivo study of hydralazine, a potential anti-angiogenic agent.</a> Eur. J. Pharmacol. 779, 138-146 (2016).</li></ol>

The authors have no competing financial interests.

En los estudios que se centran en los EC canina de la línea primaria CnAOEC se utiliza para modelar los linajes endoteliales del perro 3, 12, 13. En estudios en humanos, la cultura HUVEC todavía se considera el estándar de oro. Claramente, centrándose simplemente en ECs derivadas de cordón umbilical es una restricción firme en la investigación cardiovascular. Las células endoteliales tienen un patrón de expresión de genes específicos para determinar la especificación arteriovenosa. Con el fin de d...

Los perros se utilizan como gran modelo animal para la investigación de enfermedades cardiovasculares y también pueden sufrir de anomalías congénitas vasculares (genéticos) 1, 2. Para estudiar estas enfermedades líneas de células endoteliales comerciales a menudo se utilizan para evaluar la funcionalidad de las células endoteliales (CE). Para los perros hay una línea de células endoteliales comercial disponible (CnAOEC), derivado de la aorta canino. Esta línea celular se utiliza sobre todo en los estudios como el control normal de los EC 3-5. En la investigación cardiovascular humano las líneas de células endoteliales más comúnmente utilizadas son células endoteliales de vena umbilical humana (HUVECs) y de la arteria umbilical humana Las células endoteliales (HUAECs) derivadas de la vena umbilical humana espinal y la arteria, respectivamente. HUVECs se han utilizado como el estándar de oro en la investigación vascular desde la década de 1980 6. Ellos son considerados como el sistema clásico modelo para estudiar la función endotelial y la adaptación de la enfermedad. Las células endoteliales aisladas de diferentes vasos sanguíneos varían en appearance y funcionalidad debido a antecedentes genéticos y la exposición al microentorno 7. Además, HUVECs y HUAECs se derivan de cordón umbilical, una estructura vascular del desarrollo que no pueden vasos sanguíneos adulto completamente imitan con respecto a las condiciones que se exponen a y la respuesta a la enfermedad. Por lo tanto, la traducción de los resultados encontrados en las HUVEC y HUAECs de enfermedades cardiovasculares en general es insuficiente. Cuando se estudia la adaptación y comportamiento de los adultos EC, EC primarias del vaso de interés deben utilizarse como un enfoque más directo. Para aislar estas células, se han descrito varios métodos. Un método ampliamente descrito, que también se utiliza para HUVECs, está vaciando el recipiente con una solución de digestión enzimática 8. Esto a menudo resulta en la contaminación con no ECs tales como las células de músculo liso y fibroblastos 9. Otro método utilizado frecuentemente para el aislamiento es la digestión enzimática de tejido de vasos picada seguido de por fluorescenciade células activadas (FACS) basado en Cluster marcador de células endoteliales de diferenciación (CD) 31 7, 8. FACS clasificación y cultivo celular posterior requiere cantidades relativamente grandes de las células y por lo tanto no es adecuado para el aislamiento de endotelio de los vasos sanguíneos pequeños. Por lo tanto, el objetivo fue desarrollar un nuevo método robusto para aislar una población pura de células endoteliales de diversos vasos sanguíneos caninos con alta pureza. Para probar la eficacia del nuevo método de aislamiento, se aislaron y se obtuvieron cultivos puros Canine primaria de células endoteliales (CAPEC) de diferentes arterias y venas canino, tanto grandes como pequeñas. Este método también permite que el cultivo de células endoteliales procedentes de enfermo y / o vasos aberrantes tales como derivaciones portosistémicas intra o extra-hepáticos congénitos, una enfermedad común en los perros 2. El método permite el aislamiento de tipos de células relevantes adicionales de la misma embarcación, tales como células de músculo liso vascular ya que la mayoría de la embarcación permanece intactuar durante el procedimiento.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
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			<td style="width:201px;"></td>
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			<td height="21" style="height:21px;">Dispase</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:211px;">17105-041</td>
			<td></td>
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			<td height="21" style="height:21px;width:328px;">DMEM (1X) + GlutaMAX</td>
			<td style="width:164px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:328px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:164px;">Life Technologies</td>
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			<td style="width:164px;">Cell Applications</td>
			<td style="width:211px;">Cn211-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:328px;">CnAOECs</td>
			<td style="width:164px;">Cell Applications</td>
			<td>Cn304-05</td>
			<td></td>
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			<td style="width:164px;">GE Healthcare</td>
			<td style="width:211px;">16000-044</td>
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			<td style="width:164px;">Life Technologies</td>
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			<td height="21" style="height:21px;width:328px;">SPR</td>
			<td style="width:164px;">Bio-Rad</td>
			<td>170-8898</td>
			<td></td>
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			<td height="21" style="height:21px;width:328px;">iScript synthesis kit</td>
			<td style="width:164px;">Bio-Rad</td>
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			<td height="21" style="height:21px;width:328px;">Recovery Cell Freezing Medium</td>
			<td style="width:164px;">Gibco/Life Technologies</td>
			<td style="width:211px;">12648-010</td>
			<td>Keep on ice prior to use</td>
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			<td height="21" style="height:21px;width:328px;">Freezing container, Nalgene Mr. Frosty</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">C1562</td>
			<td></td>
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			<td height="21" style="height:21px;width:328px;">Gelatin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:211px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Surgical scissors (Mayo or Metzenbaum)</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">BC555R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps&#160;</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB440R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Mosquito forceps curved</td>
			<td style="width:164px;">B. Braun Medical</td>
			<td style="width:211px;">FB441R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">polyglactin 3-0</td>
			<td style="width:164px;">Ethicon</td>
			<td style="width:211px;">VCP311H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trypan blue</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated counting chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Counting Slides, Dual Chamber</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:211px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Matrigel</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:211px;">BD356231</td>
			<td>Slowly thaw on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#181;-Slide Angiogenesis</td>
			<td style="width:164px;">Ibidi</td>
			<td style="width:211px;">81501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Endothelial Growth Medium</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EGM-2 SingleQuot Kit&#160;</td>
			<td style="width:164px;">Lonza</td>
			<td style="width:211px;">CC-4176</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of primary endothelial cells from canine arteries and veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Diferentes vasos sanguíneos se sometieron con éxito para el protocolo de aislamiento descrito (Figura 2). Fue posible diseccionar e invierta aorta, vena cava, la vena porta y la arteria coronaria de perros sanos (todos los buques de cada perro, n = 4). Con los mismos ECs de aproximación se aislaron a partir de dos derivación portosistémica congénita (extrahepática y intrahepática, n = 1 cada uno). Aunque aorta se invierte fácilmente, los segmentos de aorta torá...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Aislamiento y cultivo de células endoteliales primarias de las arterias y venas caninas

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

isolation-culture-primary-endothelial-cells-from-canine-arteries

Research

JoVE Journal

Medicine

11.2K vistas.  Utrecht University. Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Video: Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Investigación

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultivo primario de Human vestibular schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para la obtención de las células del cáncer de ovario primarios a partir de muestras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

El aislamiento, la criopreservación y cultivo de células epiteliales Amnion Humanos para Aplicaciones clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

El aislamiento y la propagación de células tumorales circulantes de un Cáncer de ratón

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Aislamiento y cultivo de expansión de las células endoteliales-tumorales específicas

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Aislamiento de células progenitoras endoteliales de voluntarios sanos y su potencial migratorio influenciado por las muestras de suero después de cirugía cardiaca

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

Un modelo de cultivo celular de las arterias de resistencia

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...