This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Aislamiento de la aorta de los ratones Todos los procedimientos descritos aquí fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad Estatal de Wayne. <ol><li> Poner el ratón en la cámara de inducción de la máquina de anestesia. Ajuste el flujo de isoflurano al 4% en relación con el 25% de aire fresco y 75% de O 2. Anestesiar al ratón por inhalación de isoflurano (4% para la inducción, ± 1,0% para el mantenimiento) en combinación con 25% de aire fresco y 75% de O 2 a través de la cámara de inducción seguida de un cono de nariz dedicado. NOTA: La anestesia con isoflurano se puede entregar en 75% de aire de oxígeno / 25% o en el 100% de oxígeno. </li><li> Verifique el ratón cada 5 minutos hasta que el nivel adecuado de anestesia se alcanza (falta de retirada de pizca dedo del pie). </li><li> Tome el ratón fuera de la cámara de inducción y continuar la inhalación de isoflurano pesar de que el cono de la nariz dedicado que está conectado a la máquina de anestesia. conmutar elisoflurano fluya a 1% para mantener la anestesia. </li><li> Si bien es bajo anestesia, poner el ratón sobre un bloque de la cirugía, situado en su parte posterior. Use cinta de laboratorio para asegurar las extremidades. </li><li> Utilice una lámpara de calentamiento para mantener caliente el ratón. Mantenga la lámpara en una distancia apropiada del ratón de manera que el ratón no se sobre-calor. </li><li> Pulverizar el pecho con un 70% de etanol. </li><li> Use las tijeras de disección para abrir el abdomen de la línea media, y exponer la aorta abdominal. Abrir la cavidad torácica y exponer el corazón y los pulmones. </li><li> Cortar la aorta abdominal en el medio con tijeras de disección para liberar la sangre. </li><li> Llene una jeringa de 1 ml (con 25 aguja G) con 1 ml de PBS que contenía 1000 U / ml de heparina. Inyectar PBS que contenía 1000 U / ml de heparina para el ventrículo izquierdo y la aorta perfundir. Empuje el corazón y el pulmón con pinzas en las grandes arterias hasta el lado derecho de los ratones para exponer la aorta torácica. </li><li> eliminar rápidamente la aorta torácica mediante micro-difórceps ssection y ponerlo en 1x PBS helado (estéril), y luego transportar el contenedor en una campana de flujo laminar. </li><li> Inserte una jeringa de 1 ml equipado con 25 aguja G en un extremo de la aorta, y lavar suavemente la aorta con PBS enfriado en hielo para eliminar la sangre. </li><li> El uso de fórceps micro-disección para eliminar la mayor cantidad de tejido adiposo adjunto y pequeños vasos laterales como sea posible. </li><li> transferir inmediatamente la aorta endoteliales medio de crecimiento. </li><li> Cortar la aorta en 1 anillos mm utilizando una hoja de bisturí estéril. Cosecha cerca de 8 - 10 anillos por aorta. </li><li> Abrir cada anillo aórtico con un par de tijeras de microdisección. </li></ol> 2. Las semillas de los segmentos aórticos en Matrix <ol><li> Cuando no esté en uso, mantenga la matriz reducida del factor de crecimiento de los -20 ° C para evitar la solidificación. Ponga la matriz reducida del factor de crecimiento de 4 ° C al menos durante la noche para permitir que un deshielo completa. </li><li> Pre-enfriar unos 6 pocillos de placas y consejos de pipeta a -20 ° C durante al menos10 minutos. Poner la placa de 6 pocillos en hielo y capa de un pocillo de la placa con 1 ml de matriz sin introducir burbujas de aire. Colocar la placa en una incubadora a 37ºC durante 20 min para permitir que la matriz se solidifique. </li><li> Implantar las piezas de la aorta en la matriz solidificada con unas pinzas de disección micro-estériles. Colocar las piezas lumen del lado de abajo en la matriz sin tocar el endotelio. Coloque 3 - 4 segmentos aórticos cerca uno del otro en la matriz. </li><li> Añadir la cantidad necesaria medio de crecimiento de células endoteliales para mantener húmeda segmentos (~ 200 l). </li><li> Incubar la placa a 37 ° C en 5% de CO2 durante 4 a 6 h. Al final del día, añadir suficiente medio para cubrir los segmentos aórticos. </li><li> Observe el segmento aórtico brotación periódicamente bajo un microscopio de contraste de fase. Comprobar el nivel de mediano y crecimiento de las células cada día. Se añade medio si es necesario para mantener los segmentos aórticos cubiertos. </li><li> En el 4º día, retire suavemente el medio y eliminar los segmentos de aorta fesde la matriz usando una aguja estéril sin interrumpir las células endoteliales en crecimiento. </li><li> Añadir 2 ml de nuevo medio de crecimiento de células endoteliales y permitir que las células endoteliales siguen proliferando en la matriz durante 2 - 3 días. </li></ol> 3. pases inicial de las células endoteliales aórticas de ratón <ol><li> Escudo un nuevo matraz T12.5 con gelatina (0,1%), se incuba a 37 ° C durante 30 min. </li><li> Lavar las placas de matriz estéril cuidadosamente con 37 ° C de PBS. </li><li> Añadir 2 ml de proteinasa neutra (50 U / ml) a las placas de matriz y se incuba a temperatura ambiente en eje de balancín plataforma con agitación ocasional. Compruebe las células bajo un microscopio de contraste de fase para asegurarse de que la mayoría de las células se separan. </li><li> Añadir 2 ml de D-Val para inactivar la proteinasa neutra. </li><li> Recoger el sobrenadante cuidadosamente en un tubo de centrífuga de 15 mL. Lavar la placa con 2 ml de D-Val y recoger el sobrenadante. </li><li> Centrifugar la suspensión celular a 900 xg durante 5 min a Roola temperatura m. </li><li> Vuelva a suspender el sedimento de células en 4 ml de medio de crecimiento de células endoteliales y la placa en el matraz T12.5 recubierto con gelatina. Se incuban las células a 37 ° C, 5% de CO2 durante 2 h. </li><li> Reemplazar el medio. Se incuban las células a 37 ° C, 5% de CO 2 hasta que son 85% - 90% de confluencia. </li></ol> 4. pases de las células endoteliales aórticas de ratón <ol><li> Coat dos nuevos matraces T12.5 con gelatina (0,1%), se incuba a 37 ° C durante 30 min. Pre-calor tripsina-EDTA (0,25% de tripsina, EDTA 0,02 en PBS) y PBS estéril a 37 ° C durante aproximadamente 15 min. </li><li> Lavar las células cuidadosamente con estéril PBS 37 ° C. </li><li> Añadir 0,5 ml de tripsina-EDTA (0,25% de tripsina, EDTA 0,02 en PBS) y se incuba a 37 ° C durante ~ 1 min o hasta que la mayoría de las células darse la vuelta. </li><li> Añadir 2 ml de medio de crecimiento de células endoteliales para detener la digestión. Pipetear la suspensión de células arriba y hacia abajo dentro de los tiempos matraz varios. Si es necesario, utilice una célularaspador para recoger las células. </li><li> Recoger la suspensión celular en un tubo de centrífuga de 15 mL. Centrifugar la suspensión celular a 900 xg durante 5 min a temperatura ambiente. </li><li> Vuelva a suspender el sedimento de células en 4 ml de medio de crecimiento de células endoteliales y la placa en 2 frascos T12.5 recubiertas con gelatina. Se incuban las células a 37 ° C, 5% de CO2 durante 2 h. </li><li> Reemplazar el medio. Se incuban las células a 37 ° C, 5% de CO 2 hasta que son 85% - 90% de confluencia. </li><li> Use las células endoteliales de aorta de ratón después de 2 - 3 pasajes. </li></ol> 5. Caracterización de las células endoteliales de aorta de ratón <ol><li> Re-placa de las células. <ol><li> Escudo una placa de 6 pocillos con gelatina (0,1%), se incuba a 37 ° C durante 30 min. Aclarar la placa con PBS. </li><li> Pre-calentar la tripsina-EDTA y PBS estéril a 37 ° C durante aproximadamente 15 min. </li><li> Se lavan las células con PBS estéril 3 veces. Añadir 0,5 ml de tripsina / EDTA a las células, se incuba durante ~ 1 min a 37 ° C or hasta que la mayoría de las células de darse la vuelta. </li><li> Añadir 2 ml de medio de crecimiento de células endoteliales para detener la digestión. Pipetear la suspensión de células arriba y hacia abajo dentro de los tiempos matraz varios. Si es necesario, utilizar un rascador de células para recoger las células. </li><li> Recoger la suspensión celular en un tubo de centrífuga de 15 mL. Centrifugar la suspensión celular a 900 xg durante 5 min a temperatura ambiente. </li><li> Descartar el sobrenadante, volver a suspender las células en medio de crecimiento de las células endoteliales. Sembrar las células en placas de 6 pocillos recubiertas con gelatina a la densidad de 3 x 10 5 / pocillo. </li><li> Se incuban las células durante la noche a 37 ° C, 5% de CO 2 para permitir que las células se unan a la superficie de cultivo. </li></ol></li><li> Dil-ac-LDL y especialmente captado Ulex -lectin vinculante. <ol><li> Almacenar el 1,1&#39;-dioctadecil-3,3,3 &#39;, 3&#39;-tetramethylindo carbocyanine perclorato marcado LDL acetilada (Dil-ac-LDL) a -20 ° C. Antes de la tinción, descongelar Dil-ac-LDL en hielo. La solu Stock Dil-ac-LDLción es 1 mg / mL. Almacenar la aglutinina marcada con FITC Ulex europaeus (Ulex -Lectin, 1 mg / ml) a 4 ° C. La solución madre Ulex -Lectin es 1 mg / mL. Tienda Hoechst 33342 a 4 ° C. La solución madre Hoechst 33342 es 100 mg / ml. </li><li> Añadir 10 l de Dil-ac-LDL solución madre a 1 ml de medio de cultivo para hacer una concentración final de 10 mg / ml. </li><li> Antes de la tinción, se elimina el medio viejo y se lavan las células en cada pocillo con medio nuevo. </li><li> Retire el nuevo medio y se añade el medio de tinción Dil-ac-LDL. </li><li> Se incuban las células a 37 ° C, 5% de CO2 durante 1 h. </li><li> Se lavan las células con PBS 1x estéril 3 veces. </li><li> Añadir 1 ml de formalina al 10% para fijar las células. Poner la placa en la temperatura ambiente durante aproximadamente 30 min. </li><li> Se lavan las células con PBS 1x estéril 3 veces. Evitar la luz. </li><li> Añadir 1 ml de PBS 1x estéril que contiene 10 l de Ulex -Lectin. </li><li> Se incuban las células a temperatu de la habitaciónvolver en la oscuridad durante 1 h. </li><li> Retire el Ulex-lectina por lavado de las células con PBS 1x estéril 3 veces. </li><li> Añadir Hoechst 33342 a concentración final de 1 mg / ml, se incuban 10 min en la oscuridad. </li><li> Ver la fluorescencia de Dil-ac-LDL (rojo, longitud de onda de excitación de 576 nm), Ulex -Lectin (verde, la excitación de longitud de onda de 519 nm) y Hoechst (azul, longitud de onda de excitación de 361 nm) con un microscopio de fluorescencia invertida. </li></ol></li><li> La tinción fluorescente para CD31, VEGFR2, eNOS, VE-cadherina y calponin. <ol><li> Tienda FITC-conjugado anticuerpo anti-ratón CD31, anticuerpo eNOS, VE-cadherina anticuerpo, conjugado con FITC IgG anti-conejo a 4 ° C en la oscuridad. Tienda de anticuerpos VEGFR2 en -20 ° C. </li><li> Se lavan las células con PBS 1x estéril 3 veces. </li><li> Añadir 1 ml de formalina al 10% para fijar las células. Poner la placa en la temperatura ambiente durante aproximadamente 30 min. </li><li> Se lavan las células con PBS 1x estéril 3 veces. </li><li> Para la tinción de CD31, añadir 1 ml de 1x estérilPBS que contiene 10 l de anticuerpo CD31-FITC. Para teñir ya sea VEGFR2, eNOS, VE-cadherina o calponin, agregar 1 ml de PBS 1x estéril que contiene 4 l de VEGFR2, eNOS, VE-cadherina o calponin anticuerpo primario, respectivamente. </li><li> Se incuban las células en hielo durante 1 h en la oscuridad. </li><li> Eliminar los anticuerpos por lavado de las células con PBS estéril 3 veces. </li><li> Para la tinción de VEGFR2, eNOS, VE-cadherina o calponin, añadir 1 ml de PBS 1x estéril que contiene 10 l de IgG anti-conejo conjugado con FITC. Se incuban las células en hielo durante 1 h en la oscuridad. Eliminar la IgG por lavado de las células con PBS estéril 3 veces. </li><li> Añadir Hoechst 33342 a concentración final de 1 mg / ml, se incuban 10 min en la oscuridad. </li><li> Ver la fluorescencia de CD31, VEGFR2, eNOS, VE-cadherina, calponin (verde, longitud de onda de excitación de 518 a 535 nm) y Hoechst (azul, longitud de onda de excitación de 361 nm) con un microscopio de fluorescencia invertida. </li></ol></li></ol>

<ol>
	<li>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=Implications+of+early+structural-functional+changes+in+the+endothelium+for+vascular+disease.">Implications of early structural-functional changes in the endothelium for vascular disease.</a> Arteriosclerosis, thrombosis, and vascular biology. 27, 266-274 (2007).</li><li>Cid, M. C., Segarra, M., Garcia-Martinez, A., Hernandez-Rodriguez, J. <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,+antineutrophil+cytoplasmic+antibodies,+and+cytokines+in+the+pathogenesis+of+systemic+vasculitis.">Endothelial cells, antineutrophil cytoplasmic antibodies, and cytokines in the pathogenesis of systemic vasculitis.</a> Current rheumatology reports. 6, 184-194 (2004).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Reactive+protein-induced+endothelial+microparticle+generation+in+HUVECs+is+related+to+BH4-dependent+NO+formation.">C-Reactive protein-induced endothelial microparticle generation in HUVECs is related to BH4-dependent NO formation.</a> Journal of vascular research. 44, 241-248 (2007).</li><li>Wang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+circulating+CD31+/CD42-+microparticles+are+associated+with+impaired+systemic+artery+elasticity+in+healthy+subjects.">Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects.</a> American journal of hypertension. 20, 957-964 (2007).</li><li>Mackay, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterisation+of+human+pulmonary+microvascular+endothelial+cells+from+patients+with+severe+emphysema.">Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema.</a> Respiratory research. 14, 23 (2013).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> Journal of immunological methods. 244, 205-215 (2000).</li><li>Bowden, R. 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=Role+of+alpha4+integrin+and+VCAM-1+in+CD18-independent+neutrophil+migration+across+mouse+cardiac+endothelium.">Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.</a> Circulation research. 90, 562-569 (2002).</li><li>Lu, Y., 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=Palmitate+induces+apoptosis+in+mouse+aortic+endothelial+cells+and+endothelial+dysfunction+in+mice+fed+high-calorie+and+high-cholesterol+diets.">Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets.</a> Life sciences. 92, 1165-1173 (2013).</li><li>Atkins, G. B., Jain, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+differentiation:+molecular+mechanisms+of+specification+and+heterogeneity.">Endothelial differentiation: molecular mechanisms of specification and heterogeneity.</a> Arteriosclerosis, thrombosis, and vascular biology. 31, 1476-1484 (2011).</li><li>Aitsebaomo, J., Portbury, A. L., Schisler, J. C., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brothers+and+sisters:+molecular+insights+into+arterial-venous+heterogeneity.">Brothers and sisters: molecular insights into arterial-venous heterogeneity.</a> Circulation research. 108, 929-939 (2008).</li><li>Shimamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+and+foods+on+contraction+of+endothelial+cells+as+a+key+mechanism+in+atherogenesis+and+treatment+of+atherosclerosis+with+endothelial-cell+relaxants+(cyclic+AMP+phosphodiesterase+inhibitors).">Drugs and foods on contraction of endothelial cells as a key mechanism in atherogenesis and treatment of atherosclerosis with endothelial-cell relaxants (cyclic AMP phosphodiesterase inhibitors).</a> Advances in experimental medicine and biology. 60, 77-105 (1975).</li><li>Chiu, J. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+disturbed+flow+on+vascular+endothelium:+pathophysiological+basis+and+clinical+perspectives.">Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives.</a> Physiological reviews. 91, 327-387 (2011).</li><li>Gimbrone, M. A., Topper, J. N., Nagel, T., Anderson, K. R., Garcia-Cardena, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction,+hemodynamic+forces,+and+atherogenesis.">Endothelial dysfunction, hemodynamic forces, and atherogenesis.</a> Annals of the New York Academy of Sciences. 902, 239-240 (2000).</li><li>Rostgaard, J., Qvortrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+demonstrations+of+filamentous+molecular+sieve+plugs+in+capillary+fenestrae.">Electron microscopic demonstrations of filamentous molecular sieve plugs in capillary fenestrae.</a> Microvascular research. 53, 1-13 (1997).</li><li>Brutsaert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial-myocardial+signaling:+its+role+in+cardiac+growth,+contractile+performance,+and+rhythmicity.">Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity.</a> Physiological reviews. 83, 59-115 (2003).</li></ol>

The authors have nothing to disclose.

Este estudio demuestra un método simple para aislar y endotelial cultura células de una aorta de ratón sin ningún equipo especial. La tinción de inmunofluorescencia indicó que la mayoría de las células eran células endoteliales después de la segunda pasaje. Se sugiere que las células en segundo-tercio paso son apropiados para in vitro e in vivo para estudiar la biología endotelial. Las Notas clave de la presente Protocolo <p class="jove_cont...

El endotelio vascular es no sólo una capa de barrera que separa la sangre y el tejido, se considera una amplia glándula endocrina que se extiende sobre todo el árbol vascular con una superficie de 400 metros cuadrados 1. El bienestar del endotelio es esencial para la homeostasis vascular. El endotelio disfuncional participa en diversos trastornos cardiovasculares, incluyendo la aterosclerosis, vasculitis y las lesiones por isquemia / reperfusión, etc. 2-4. Hasta la fecha, los mecanismos celulares y moleculares específicos implicados en estos ajustes de la enfermedad no se comprenden bien debido a la naturaleza anatómica difusa de endotelio. El ratón es un modelo importante para la investigación porque las técnicas de manipulación genética están más desarrollados en ratones que en cualquier otra especie de mamífero. Sin embargo, el aislamiento de células endoteliales aórticas primaria murino se considera particularmente difícil debido a que el pequeño tamaño de la aorta hace enzymatdigestión ic de endotelio poco práctico. Algunos informaron procedimientos para aislar y purificar las CE exijan 5-7. El objetivo de este protocolo es el uso de un método simple para aislar y expandir las células endoteliales de la aorta de ratón sin utilizar ningún equipo especial. En este protocolo, la aorta recién aisladas se corta en pequeños segmentos y se sembró sobre una matriz con el endotelio hacia abajo para permitir la germinación endotelial. Después de segmentos se eliminan, las células endoteliales se expandieron en medio del endotelio favorecida y están listas para experimentos después de dos o tres pasajes. Las ventajas del método descrito son que: 1) considerablemente alto número de células endoteliales se obtienen de una sola aorta; 2) la viabilidad celular está bien conservada; y 3) no se necesita ningún equipo o técnica especial. Se proporciona un medio eficaz para la identificación de los mecanismos celulares y moleculares específicos en la fisiopatología de las células endoteliales. Para aquellos que estén interesados ​​en el estudio de prLas células endoteliales cultivadas imary de cualquiera de los genes de ratones knock-out, genes knock-en ratones, o un modelo murino de la enfermedad, este protocolo es muy útil y fácil de practicar.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

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.

Brote de la célula endotelial espontánea de las células endoteliales brotación comenzó a partir de un segmento de la aorta del ratón. El segmento de aorta de ratón se dejó crecer en una matriz reducida del factor de crecimiento a continuación en medio de crecimiento de células endoteliales durante 4 días. El surgimiento de las células endoteliales por lo general aparece en 2 - 4 días. Las microfotografías se tomaron el día 4 (Figura 1). Como s...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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-and-primary-culture-of-mouse-aortic-endothelial-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

30.4K vistas.  Wayne State University. 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.

Video: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

Aislamiento de células primaria murino cerebro endoteliales microvasculares

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Investigación

Neurociencias

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 ...

Biología

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 ...

Medicina

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Aislamiento de las células endoteliales microvasculares primaria murinas músculo esquelético

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

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 Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Aislamiento de células endoteliales pulmonares primarias de ratón

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Inmunología e Infección

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Aislamiento de murino coronaria células musculares lisas vasculares

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Aislamiento y caracterización de células endoteliales sinusoidales sinusoidales hepáticas primarias de ratón

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...