Esta investigación fue financiada en parte por la Asociación Americana del Corazón conceder 0950118G.to MC-W.

1. La preparación de partículas magnéticas anti-PECAM-1 anticuerpo conjugado (Dynabeads) <ol><li> Prepare un 0,1% de BSA en PBS mediante la mezcla de 50 mg de BSA en 50 ml de PBS con un agitar hasta BSA se disuelve. Esterilizar por filtración a través de filtro de 0,22 micras jeringa. Almacenar a 4 ° C. </li><li> En la capilla de cooperación técnica, transferencia de 200 l de bien resuspendido de oveja anti-ratón IgG Dynabeads en un tubo Eppendorf de 1,5 ml y lavar los granos: el tubo de montaje en la MPC y deje reposar durante 1 minuto para permitir que los granos de sedimento. Aspirar el sobrenadante, retire el tubo de MPC y volver a suspender las bolas en 1 ml de agua estéril 0,1% de BSA / PBS. Pipeta de arriba y abajo para volver a suspender las bolas. </li><li> Repetir el lavado tres veces más, para un total de cuatro lavados. </li><li> Sacar el tubo del MPC y volver a suspender las bolas en 500 l de 0,1% de BSA / PBS. </li><li> Añadir 10 l de rata anti-ratón de PECAM-1 (CD-31) del anticuerpo en el tubo. </li><li> Caída la noche a 4 ° C en la cámara frigorífica o 2 horas a temperatura ambiente. </li><li> Lavar los granos con agua estéril 0,1% de BSA / PBS como se describe en el paso 1.2 en cuatro ocasiones. </li><li> Sacar el tubo del MPC y volver a suspender las bolas en 200 l 0,1% de BSA / PBS. Tienda anti-PECAM-1 anticuerpo conjugado con Dynabeads a 4 ° C durante un máximo de un mes. </li></ol> 2. Aislamiento de ratón las células endoteliales pulmonares de ratones recién nacidos Antes de proceder con el protocolo de purificación de los tejidos, IACUC aprobación del Comité del procedimiento es necesario. <ol><li> Prepare las siguientes: <ul><li> 15 ml de 1 mg / ml C / D solución en DMEM. Filtro con filtro de 0,22 micras jeringa y calentar a 37 º C. </li><li> Tubo de 50 ml cónicos con 15 ml de DMEM, almacenar en el hielo </li><li> Aislamiento de los medios de comunicación (mensajería instantánea), que contiene 20% de SFB y la penicilina 1x / estreptomicina en DMEM </li><li> 2% de gelatina. Mezcla de 2 g de piel de bovino de gelatina con 100 ml de agua dd, minuto 15 autoclave y almacenar a 4 ° C. Calentar a 37 ° C para licuar antes de pintar frascos TC. </li></ul></li><li> Anestesiar a tres crías de 6-8 días de edad con una inyección ip de ketamina (100 mg / kg) y xilazina (10 mg / kg). Comprobar la efectividad de la anestesia y la disección de los animales uno por uno, de la siguiente manera: Pin cachorros a bordo, empape la piel con etanol 70% y eliminar la piel de la zona del pecho con unas tijeras de disección estéril. Impuestos Especiales de la caja torácica para exponer los pulmones. </li><li> Lóbulos individuales asépticamente especial de pulmón, teniendo cuidado de no analizar los bronquios y los tejidos conectivos visible alrededor de los pulmones. Combine todos los pulmones en helado de DMEM. La eutanasia a los cachorros. </li><li> De trabajo en la campana TC, remueva los lóbulos pulmonares de DMEM, el lugar en un plato de 100 mm TC y aspirar el exceso de DMEM. </li><li> Utilizando tijeras estériles, carne picada finamente el tejido mediante la reducción de ~ 100 veces. Traslado a 15 ml caliente C / D solución para la digestión enzimática. Incubar 45 minutos a 37 ° C en un rotor. </li><li> En la capilla TC, aspirar la suspensión de tejidos digeridos en una jeringa de 20 ml con 14 g grupos cánula unida y triturar en una suspensión de células individuales, por lo menos 12 veces. </li><li> Pase la suspensión de tejido a través de un filtro de 70 micras de células y lavar el filtro con 15 ml de mensajería instantánea para detener la digestión. </li><li> Pellet suspensión de células por centrifugación a 400x g durante 5 minutos. </li><li> Aspirar el sobrenadante y el precipitado se resuspende en 3 ml de 0,1% de BSA / PBS. </li><li> Transferencia de suspensión de células a 5 ml tubo de poliestireno de fondo redondo y añadir 22,5 l anti-PECAM-1 anticuerpo conjugado-Dynabeads. Caída en la temperatura ambiente durante 12 minutos. </li><li> Cubra un frasco T75 con 2 ml de 2% de gelatina y el exceso de aspirado de gelatina. Permita que la gelatina seca en su interior la capilla TC durante unos 30 minutos antes de las células de revestimiento. </li><li> Después de la incubación de cuentas es completa (paso 2,10), dividir la suspensión de células por igual en tres tubos Eppendorf y se montan en el MPC. </li><li> Cuando las cuentas se han sedimentado con el MPC (~ 1 minuto), recoger el líquido sobrenadante, retire el tubo de la MPC, lavar los granos con aproximadamente 1 ml 0,1% de BSA / PBS, y volver a montar en el MPC. </li><li> Repetir el lavado cuatro veces (un total de 5 lavados) </li><li> Cuentas resuspender en 1 ml de VascuLife con ENG-Mv vida Factores de Kit y 1 de penicilina / estreptomicina (VL) por tubo, se mezcla bien. </li><li> Placa en una pre-recubiertos T75 frasco y alcanzar un volumen total en cada frasco de 10 ml a la LV. </li><li> Cambiar los medios de comunicación al día siguiente, deje un día lleno de crecimiento, y luego cambiar la mitad de los medios de comunicación todos los días. Cuando las células son&gt; 70-80% confluentes o más (por lo general después de 3-4 días de la cultura), que pueden ser ordenados con anti-ICAM-2 Dynabeads. </li></ol> 3. La preparación de anti-ICAM-2-anticuerpo conjugado Dynabeads <ol><li> Dynabeads anti-ICAM-2-anticuerpo conjugado se utiliza para purificar las células que han sido seleccionados con anti-PECAM-1 Dynabeads anticuerpos conjugados y cultivaron hasta confluencia. Este procedimiento se realiza como se describe para Dynabeads anti-PECAM-1 anticuerpo conjugado (1, arriba), excepto que el anti-ratón ICAM-2 (CD-102) del anticuerpo se utiliza en lugar de anti-PECAM-1 anticuerpo. </li><li> Dynabeads anti-ICAM-2-anticuerpo conjugado se puede almacenar a 4 ° C hastaa 1 mes. </li></ol> 4. Clasificación de las células de ratón de pulmón endoteliales con anticuerpos anti-ICAM-2 Dynabeads <ol><li> Escudo T75 frasco de cultivo de tejidos con gelatina al 2%. </li><li> Aspirar los medios de comunicación de la confluencia con el T75 frasco con células y lavar con 8 ml de PBS. </li><li> Aspirar PBS, añadir 2 ml al 0,05% de tripsina / EDTA y dejar que las células de quitar (unos 5 minutos). Toque en frascos ligeramente para ayudar a separar. </li><li> Cuando las células se han desprendido, añadir 2 ml IM para inhibir las células de la tripsina y la raspadura de separar las células adherentes restantes. Transferencia de suspensión de células a un tubo de 15 ml y centrifugar durante 5 minutos a 400 veces g. </li><li> Aspirar los medios de comunicación y resuspender el sedimento celular en 2 ml de 0,1% de BSA / PBS. </li><li> Transferir a un 5 ml de poliestireno de fondo redondo de tubo y se añaden 10 l anti-ICAM-2 Dynabeads. Caída por 12 minutos a temperatura ambiente. </li><li> Dividir la suspensión de células en dos tubos de 1,5 ml Eppendorf y se montan en MPC. </li><li> Aspirar el sobrenadante después de cuentas se han adherido durante un minuto. Retire los tubos del MPC y resuspender en cada tubo 1 ml 0,1% de BSA / PBS. Vuelva a colocar el MPC. </li><li> Repetir el lavado cuatro veces (un total de 5 lavados). </li><li> Resuspender cada tubo con 1 ml VL y realizar un recuento de células. </li><li> Células de la placa a 250-350K células por frasco T25 y / o 750K-1 millón de células por frasco T75. </li><li> Alcanzar un volumen de 5 ml VL en frascos T25, y 10 ml en frascos T75. </li><li> Cambiar los medios de comunicación todos los días. Las células pueden ser utilizados para los experimentos en que la cultura llega a cerca del 80% de confluencia, por lo general en 2-3 días. </li></ol> 5. Resultados representante Normalmente, después de 6-7 días a partir de la preparación inicial de las células, que son capaces de obtener unos 1,2 a 1,5 x 10 6 células endoteliales pulmonares de tres crías de 6-8 días de edad. Células típicos de la pantalla &quot;adoquines&quot; morfología con microscopía de luz y VE-cadherina (CD144) manchas en las uniones célula-célula, que es característico de las células endoteliales (Figura 1). La mayoría de los MLECs expresa VE-cadherina y VEGFR2 como lo demuestra el análisis de FACS (Figura 2). Por lo general, los utilizamos para los experimentos dentro de dos semanas después de la inicial de aislamiento LMCE. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="Figura 1" /> Figura 1. El análisis microscópico de monocapa confluente LMCE. (A) Imagen de microscopía de luz muestra la morfología de las células cultivadas empedradas típicas de las células endoteliales. Uniforme de estructuras redondas ubicadas en la zona perinuclear de las células son muchas las partículas magnéticas utilizadas para el aislamiento LMCE. (B) endotelial específico VE-cadherina se localiza en las uniones célula-célula, como se muestra mediante microscopía confocal. Bar es representante de 20μm. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="Figura 2" /> Figura 2 FACS análisis de MLECs cultivadas con anticuerpos específicos para los marcadores específicos del endotelio:. VE-cadherina (CD144) o VEGFR2, como acusado, seguido por ficoeritrina-anticuerpo secundario conjugado, confirma la identidad del endotelio de MLECs aislados. La línea roja indica isotipo específico de control, la línea verde indica anti-VE-cadherina o anti-VEGFR2 - específicos IgG.

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <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+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <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+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <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+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+vascular+endothelial+cadherin+(7B4/cadherin-5),+an+endothelium-specific+cadherin.">Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.</a> Arterioscler Thromb Vasc Biol. 15, 1229-1239 (1995).</li><li>Zachary, I., Gliki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+transduction+mechanisms+mediating+biological+actions+of+the+vascular+endothelial+growth+factor+family.">Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family.</a> Cardiovasc Res. 49, 568-581 (2001).</li><li>Diglio, C. A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. E., Gale, D., White, G. C., Vansluys, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+angiogenesis,+endothelial+migration,+proliferation,+and+MAPK+signaling+in+Rap1b-deficient+mice.">Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice.</a> Blood. 111, 2647-2656 (2008).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y regulaciones establecidas por el Colegio Médico de Wisconsin IACUC Comité en virtud del protocolo AUA # 00001206.

Microvascular EC han demostrado ser un modelo útil en muchas áreas de la biología vascular y se cree que son fisiológicamente más relevantes para el estudio (por ejemplo, la angiogénesis) que ampliamente estudiado HUVECs 3. Anteriormente, se ha informado de que microvascular EC se puede obtener de los riñones, el corazón, el tejido adiposo piel, retina, el cerebro, los gliomas, y también de pulmón 2, 4-7, 10, 11. Sin embargo, un método consistente y fiable para el aislamiento de complicaciones microvasculares EC sigue siendo necesaria. El procedimiento que aquí se presenta es una modificación de un protocolo previamente publicado 2, se diferencia de la mayoría de los procedimientos publicados, ya que utiliza los cachorros de animales adultos. Esto es crucial para el éxito de aislamiento como las células de los animales jóvenes tienen un potencial mayor proliferación y cultivos derivados de los tejidos tienden a producir un mayor número de células. Una semana de edad los animales parecen ser óptimo, pero ratones más jóvenes (de un día 4), también se puede utilizar. Además, el número mayor o menor de las crías puede ser utilizado en caso necesario, como hemos tenido éxito en aislar MLECs de un cachorro. Sin embargo, mientras que la escala hacia arriba o hacia abajo el proceso de aislamiento, la densidad celular recubrimiento debe permanecer sin cambios, ya que esto también es fundamental para la proliferación celular. El proceso de purificación de 2 pasos de MLECs usando perlas magnéticas conjugadas primero en anti-PECAM-1 y de anti-ICAM-2 anticuerpos es mucho más eficiente en la obtención de cultivos puros CE de los procedimientos de purificación se ha descrito anteriormente en un solo paso. Este protocolo elimina la necesidad de utilizar técnicas manuales muy laborioso, y centrifugación en gradiente de FACS menos eficientes de ordenación de las células de descartar la contaminación como los fibroblastos, las células sanguíneas y células musculares lisas. La población resultante LMCE se puede utilizar para el análisis in vitro de las respuestas de la angiogénesis, la permeabilidad vascular y la transmigración de leucocitos, cicatrización de heridas, así como el análisis bioquímico de las vías de señalización. Si es necesario, MLECs se puede platear en diferentes superficies, tales como fibronectina cubreobjetos recubiertos de gelatina o por inmunofluorescencia o matrices de electrodos para trans-endotelial de resistencia (TER) mediciones. Resultados obtenidos se pueden comparar directamente con los fenotipos observados en vivo, que es especialmente importante en el contexto de la creciente cantidad de octavos de final y las líneas de ratones transgénicos. La implementación exitosa de este método de análisis in vitro de los mecanismos celulares subyacentes defectos observados en vivo, ya se ha presentado 12.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of pulmonary endothelial cells from neonatal mice

Las células endoteliales proporcionar un modelo de investigación útil en muchas áreas de la biología vascular. Desde su primer aislamiento 1, las células endoteliales de vena umbilical (HUVEC) han demostrado que son convenientes, fáciles de obtener y la cultura, y por lo tanto son las células más estudiadas endoteliales. Sin embargo, la investigación se centró en los procesos como los demás la angiogénesis, la permeabilidad o muchos, las células endoteliales microvasculares (EC) es un modelo mucho más fisiológicamente relevantes para el estudio 2. Por otra parte, los CE aislado de ratones knock-out proporcionar una herramienta útil para el análisis de la ex vivo función de las proteínas. Varios métodos para aislar y microvascular cultura EC de origen diferentes han sido reportados hasta la fecha 3-7, pero el aislamiento constante y la cultura de la pura EC sigue siendo un gran problema técnico en muchos laboratorios. Aquí, ofrecemos un protocolo de paso a paso en un método fiable y relativamente fácil de aislar y cultivar células de los pulmones del ratón endotelial (MLECs). En este enfoque, el tejido pulmonar obtenido a partir de 6 - a 8 días de edad los cachorros primero se corta en pedazos, digeridos con colagenasa / dispasa (C / D) y la solución dispersa mecánicamente en una sola célula de la suspensión. MLECS se purificó a partir de la suspensión celular mediante la selección positiva con anti-PECAM-1 anticuerpo conjugado con Dynabeads utilizando un concentrador de partículas magnéticas (MPC). Tales células purificadas se cultivan en cultivo de tejidos recubiertos de gelatina (TC) los platos hasta que se hacen confluentes. En ese momento, las células se purifica utilizando Dynabeads junto a anti-ICAM-2 de anticuerpos. MLECs obtenidos con este protocolo muestran un fenotipo de adoquín, como se visualiza mediante microscopía óptica de contraste de fase, y su fenotipo endotelial ha sido confirmado mediante análisis de FACS con anticuerpos anti-VE-cadherina 8 y 9 VEGFR2 anti-anticuerpos y la tinción de inmunofluorescencia de VE-cadherina. En nuestras manos, este procedimiento de aislamiento de dos etapas consistente y fiable rendimiento de una población pura de MLECs, que puede ser más culta. Este método permitirá a los investigadores para aprovechar el creciente número de eliminatorias y ratones transgénicos que se correlacionan directamente en estudios in vivo con los resultados de los experimentos in vitro realizados en MLECs aisladas y por lo tanto ayudar a revelar los mecanismos moleculares de los fenotipos vasculares observados en vivo.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Aquí se describe un protocolo para el aislamiento y cultivo de las células endoteliales murinas pulmonar. Este método consiste en la disociación mecánica y enzimática del tejido pulmonar, así como un proceso de purificación de 2 pasos con anti-PECAM-1 y anti-ICAM-2 anticuerpos conjugados a perlas magnéticas, lo que produce una población pura de células endoteliales microvasculares su mayoría de origen.

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 ...

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Research

JoVE Journal

Biology

46.9K vistas.  BloodCenter of Wisconsin. Aquí se describe un protocolo para el aislamiento y cultivo de las células endoteliales murinas pulmonar. Este método consiste en la disociación mecánica y enzimática del tejido pulmonar, así como un proceso de purificación de 2 pasos con anti-PECAM-1 y anti-ICAM-2 anticuerpos conjugados a perlas magnéticas, lo que produce una población pura de células endoteliales microvasculares su mayoría de origen.

Video: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

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

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

Aislamiento de células endoteliales valvular

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

Investigación

Biología

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

El aislamiento de la arteria pulmonar células musculares lisas de los ratones recién nacidos

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

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

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

Aislamiento de las células murinas de la válvula endoteliales

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

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

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Generación y Cultura de Sangre Consecuencia endoteliales Las células de la sangre periférica humana

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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 Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...