Name: isolation of mouse coronary endothelial cells
Uploaded: 2016-07-03T13:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Este trabajo fue apoyado por la subvención de los Institutos Nacionales de Salud (HL115578 de A. Makino).

La investigación en ratones fue aprobado por el Comité Institucional de Cuidado de Animales y el empleo (IACUC) de la Universidad de Arizona y se llevó a cabo de acuerdo con el Instituto Nacional de Salud (NIH) Directrices. NOTA: Las secciones 1, 2 y 3 deben llevarse a cabo el día antes del experimento como configuración. 1. Preparación de Soluciones <ol><li> tampón de CD31 (50 ml) <ol><li> Añadir 50 ml de 1 x tampón fosfato salino (PBS) a un tubo de 50 ml. Pesar 0,05 g Albúmina de suero bovino (BSA) y 0,037 g de ácido etilendiaminotetraacético (EDTA) y se añade al tubo de 50 ml y colocar el tubo de girar durante aproximadamente 1 h hasta que el polvo se disuelva. Ajustar el pH a 7,4, se filtra a través de un filtro de 0,2 micras en un nuevo tubo de 50 ml y se almacena a 4 ° C hasta su uso. </li></ol></li><li> Solución salina equilibrada de Hank (HBSS) con HEPES (500 ml) <ol><li> Añadir 1,1915 g de HEPES hacer HEPES 10 mM en solución HBSS y mezclar bien. Medir el pH y ajustar a 7,4. Se filtra y se almacena a 4DO. </li></ol></li><li> 1X solución de Kreb (1 L) <ol><li> Añadir 100 ml de solución de 10 × de Krebs [Tabla 1] para duplicar agua destilada en un matraz de 1 L. Pesar 2,1 g de bicarbonato de sodio y 2 g de D-glucosa y añadir al matraz. Mezclar y ajustar el volumen total a 1 L. </li><li> Solución de la burbuja con un 95% de O2 / 5% de CO2 del gas durante 30 minutos y se filtra la solución dentro de la campana. Almacenar a 4 ° C y se mantiene en hielo durante el experimento. </li></ol></li></ol> 2. Preparación de las perlas magnéticas <ol><li> Añadir 1 ml de tampón de CD31 en cada tubo 1,5 ml (un tubo por muestra). Mezclar las perlas de IgG anti-rata de oveja y añadir la cantidad apropiada para cada muestra [Tabla 1]. Agitar vigorosamente los tubos 30 veces y colocarlos en la placa magnética durante 1 min de agitación. Abrir los tubos mientras que en el plato y volcar las soluciones en un cubo. </li><li> Añadir 1 ml de tampón de CD31 en cada tubo. Añadir 2 l de rata anti-ratón anti CD31cuerpo a cada tubo y girar tubos O / N a 4 ºC. </li></ol> 3. El tratamiento en autoclave <ol><li> Autoclave todas las herramientas de cirugía y puntas de pipeta. Cortar los extremos de las puntas de pipeta de manera que hay consejos con 4 diámetros diferentes, de mayor a más estrecho. diámetros aproximados de los consejos son de 3 mm, 2,5 mm, 2 mm y 1,5 mm, respectivamente. Cortar las puntas y autoclave el día anterior con 30 minutos de esterilización a una temperatura de 121 ºC. NOTA: Secciones 4-11 deben llevarse a cabo en el día del experimento. </li></ol> 4. Preparación de Soluciones <ol><li> Preparar el tampón de lavado que contenía 2% enriquecida con hierro de suero de ternera (FISQ) en M199. Para imágenes, preparar 30 ml / ratón. </li><li> Preparar la solución de enzima (10 ml / muestra). Pesar 0,6 unidad / ml de dispasa y 1 mg / ml de colagenasa de tipo II en un tubo de 50 ml. Añadir M199 y rotar el tubo a 4 ºC durante 15 minutos. Filtrar a través de filtro de 0,2 micras en un nuevo tubo de 50 ml inside la campana y mantener a 4 ° C hasta su uso. </li><li> Preparar HBSS / HEPES con heparina. Añadir 10 ml de HBSS con HEPES 10 mM en 15 ml tubo. Añadir 100 ml de heparina al tubo y mantenerla en 4 ° C hasta su uso. </li></ol> 5. lavado de las perlas <ol><li> Sacar los tubos con los granos de la rotador a 4 ºC y llevar a la campana. Colocar los tubos en la placa magnética y agitar durante 1 min. Volcado de la solución y añadir tampón CD31 1 ml en cada tubo. </li><li> Tomar los tubos de la placa magnética y agitar vigorosamente 30 veces. Poner los tubos de nuevo en la placa magnética y repetir para un total de 3 lavados. Después de la terminación, poner los tubos de nuevo a 4 ° C y mantener girando hasta su uso. </li></ol> 6. La disección <ol><li> Inyectar los ratones por vía intraperitoneal con 0,1 ml de heparina para prevenir la coagulación de la sangre. Espere 10 minutos y, a continuación anestesiar a los ratones utilizando la inyección intraperitoneal de 0,01 ml de pentobarbital. Compruebe si el ratón está sintiendodolor por pinzamiento del pie. </li><li> Coloca el ratón sobre una placa de poliestireno con la cabeza hacia arriba y mantenga los brazos con alfileres. El uso de fórceps y duras cortadas tijeras para cortar la piel en la zona torácica superior hasta la garganta. </li><li> Hacer un corte en el centro de la caja torácica justo por encima del diafragma. Cortar el hueso hacia los lados y hacia arriba, hacia la garganta para hacer un triángulo exponer el corazón y los pulmones. Cortar la aorta justo por encima del diafragma. </li><li> Mantenga el timo con unas pinzas sin tirar y usar las tijeras para cortar cualquier tejido conectivo cuidadosamente para eliminar los pulmones y el corazón juntos. Coloque los tejidos en un vaso de precipitados con 1 × Krebs de, agitar con unas pinzas y se transfieren a placa de 6 cm con 1 × Krebs de. </li><li> Retire los lóbulos pulmonares utilizando tijeras y pinzas. Insertar un catéter estéril 20 G en la aorta. Enjuague la sangre en el sistema circulatorio coronario con HBSS / heparina. Hacer un pequeño corte en el ventrículo, agitar el corazón y transferir a un tubo de muestra de M199. Mantenga los tubos en hielo. </li></ol><p<html> class = &quot;jove_title&quot;&gt; 7. La digestión del tejido <ol><li> Transferir el corazón a una placa de 6 cm llena con 10 ml de solución de enzima y picar en trozos pequeños con tijeras. Colocar las cápsulas en la incubadora a 37 ° C durante 15 minutos. Agitar materiales digeridos por pipeteando arriba y abajo 30 veces a través de una punta con un extremo cortado para permitir que el tejido pase a través de más fácil. </li><li> Poner de nuevo en la incubadora de 37 ° C durante otros 15 minutos. Repita la agitación tres veces, cada vez utilizando puntas de pipeta de grandes aberturas para más estrecho y 15 minutos de incubación entre cada uno. </li><li> Después de la última incubación de 15 minutos, tomar muestras y en la campana. solución pipetear hacia arriba y hacia abajo 30 veces usando punta de la pipeta con la abertura más estrecha y pasar por un colador de 40 micras de células en un nuevo tubo de 50 ml. </li><li> Lavar el plato con tampón de lavado de 10 ml con una pipeta de 10 ml y transferir a células colador. Poner los tubos de 50 ml en la centrífuga y centrifugado a 400 g durante 10 min a TA. </li></ol>itle &quot;&gt; 8. Lavado y conjugación con los granos <ol><li> Extraer el sobrenadante sin perturbar el sedimento. Lavar las células con tampón de lavado 5 ml dos veces. </li><li> Consigue uno de los previamente preparados tubos de 1,5 ml con los granos y el trabajo sobre un tubo cada vez. Ponga un tubo de 1,5 ml en la placa magnética, agitar 3 veces y abrirlo. </li><li> Después de la última centrífuga, eliminar la mayor cantidad sobrenadante como sea posible sin perturbar el sedimento. Disociar los grupos de células con un movimiento rápido con vigor. </li><li> Añadir tampón de lavado 1 ml al tubo de 50 ml y mezclar las células con una pipeta de 1 ml. Volcado de la solución del tubo de 1,5 ml y se añade 1 ml de la suspensión celular del tubo de 50 ml a las perlas. Flick los tubos 30 veces y luego girar a 4 ºC durante 30 minutos. </li></ol> 9. Preparación de Cultivos Celulares / Imaging <ol><li> Preparar cámaras para el recubrimiento de las células. Añadir solución de gelatina (5% w / v, 300 l) a la cámara de bien para cubrir la superficie y se incuba durante 30 min a 37 ºC. Después de la incubación, remove gelatina y seca la superficie. </li><li> Sacar los tubos con perlas / células después de 30 minutos de rotación y poner en el plato magnético en el interior de la campana. Agitar durante 2 min, volcado y añadir tampón de lavado 1 ml. </li><li> Tome tubos fuera de la placa, agitar vigorosamente 30 veces y luego flick 30 veces. Ponerlos de nuevo en placa magnética y agitar durante 1 min. Repetir 4 veces para un total de 5 lavados, pero con 1 min agitando en el plato. </li><li> Después del último lavado, saque un tubo y trabajar en cada uno de los tubos a la vez. Agite 30 veces y luego chasquear 30 veces. Póngalo en la placa magnética y agitar durante 1 min. Volcar y se añade 1 ml de 20% de medio IFCS CE (pre-calentado a 37 ºC). </li><li> Agitar enérgicamente 30 veces y luego flick 30 veces. Ajustar la pipeta de 500 l y pipetear hacia arriba y hacia abajo 10 veces. Transferir 500 l de cada cámara. </li><li> Ponga a 37 ºC y dejar incubadora O / N. Al día siguiente, lavar las células con medio FBS 10% de la CE. </li></ol> 10. Preparación para la Blot Las muestras Western (WB) <ol><li> Take los tubos con perlas / células después de 1 h de rotación. Ponerlos en la placa magnética y agitar durante 2 min. Volcar sobrenadante y añadir 1 ml de HBSS frío. Agitar enérgicamente 30 veces y luego flick 30 veces. Repetir dos veces por 3 lavados, pero con 1 min agitando en el plato. </li><li> Tras el último lavado, poner los tubos en la placa magnética y agitar durante 1 min. Eliminar el tampón usando la pipeta y lisar las células para WB utilizando un método descrito previamente 11,12. lisado recopilada por lo general contiene alrededor de 30 g de proteína. </li></ol> 11. Imaging <ol><li> Lavar las células 3 veces con medios de comunicación, con un volumen final de 250 l para una cámara. Añadir 1,5 l de Dil-acLDL a las células. A partir de este punto, mantener las células en la oscuridad. </li><li> Incubar durante 3,5 horas a 37 ° C. Añadir 1,5 l de Hoechst, mezclar bien y se incuba durante 30 min a 37 células ˚C.Wash con PBS y fijar con 4% de paraformaldehído (PFA) durante 20 min a RT. </li><li> Lavar dos veces con PBS y bloquear con 5% BSA-PBS durante 30 min a TA. Lavar dos veces con 1% BSA-PBS. Diluir BS-lectina-FITC usando 1% BSA-PBS a una concentración de 2 mg / ml y añadir 300 l a cada cámara. </li><li> Ponga la cámara en el agitador orbital a temperatura ambiente durante 30 min. Lavar con PBS 3 veces. </li><li> Visualizar las células bajo el microscopio de fluorescencia con el tiempo de exposición de 200 ms para FITC (tinción de lectina, un marcador CE, excitación / emisión 488 nm / 519 nm), 30 ms para DAPI (Hoechst, una tinción, excitación / emisión nuclear a 358 nm / 461 nm), y 300 ms para TRITC (tinción acLDL, un marcador de la CE, excitación / emisión a 557 nm / 576 nm). Adquirir imágenes con una lente objetivo de 20X. </li></ol>

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	<li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li><li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+endothelial+cell.">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li><li>Cines, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. 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+function+and+dysfunction:+testing+and+clinical+relevance.">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li><li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <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+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung.">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li><li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice.">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</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> J Immunol Methods. 244, 205-215 (2000).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li><li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li><li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, 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+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice.">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li><li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice.">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li><li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC:+old+protein+with+new+roles+in+diabetes.">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li></ol>

The authors have nothing to disclose.

Las células endoteliales más ampliamente utilizado en la investigación son células humanas endoteliales de vena umbilical (HUVEC) debido a su comodidad y facilidad de cultivo. Sin embargo, una gran cantidad de investigación requiere la disponibilidad de un modelo fisiológico alcanzado por el aislamiento de células endoteliales de otros órganos específicos 10. Las células endoteliales forman una población muy minoritaria de las células en el tejido del corazón; su aislamiento y el cultivo puede ll...

Los modelos de ratón de diversas enfermedades cardiovasculares y los trastornos metabólicos tienen cambios fisiológicos y moleculares que son similares a los encontrados en pacientes. Por otra parte, la alteración genética de los ratones es una poderosa herramienta que nos permite investigar el papel patogénico de genes específicos en el desarrollo y progresión de enfermedades. Hoy en día, gen-knock-in célula de tipo específico o ratones salida privado se generan fácilmente en muchos laboratorios y la medición de los niveles de ARNm y de proteína en tipos específicos de células es el primer paso en la determinación de si los ratones fueron verdaderamente genéticamente modificados. El endotelio es una capa delgada única de las células endoteliales (EC) que recubre la pared vascular interior. Las células endoteliales juegan un papel crítico en la regulación de la tensión vascular, permeabilidad vascular, y la formación de nuevos vascular 1,2. La disfunción endotelial es una característica de muchas condiciones patológicas y los cambios en la función endotelial puede conducir a diversas cocheenfermedades cardiovascu- 3,4. Por tanto, es importante para estudiar la función de las células endoteliales en condiciones fisiológicas y fisiopatológicas. Hay varias maneras para aislar ECs 5-8 y el método de aislamiento tiene que ser optimizada en función de los cuales se utilizan tejidos y especies. Esto se debe a ECs de diferentes tejidos muestran un alto nivel de heterogeneidad con respecto a sus marcadores de superficie y la expresión de proteínas 9. éxito en el aislamiento de las células endoteliales a menudo puede ser difícil y requiere un cierto grado de entrenamiento y práctica. Una vez logrado, el método de aislamiento se propone en este protocolo resulta ser estable y eficiente. El objetivo general de este método es la obtención de ratón EC coronaria (MCECs) de alta calidad y la cantidad. A pesar de que la cantidad de las células recogidas de un corazón es menor que las células recogidas de otros tejidos que utilizan otras técnicas, esta técnica todavía proporciona apuestater calidad. La pureza de las células endoteliales en la población resultante es mayor que 90%. Por lo tanto, esta técnica sería ideal para aplicaciones que dependen de las células endoteliales puramente aislados, como imágenes de células.

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

isolation of mouse coronary endothelial cells

Las células endoteliales revisten la pared interna de los vasos sanguíneos y juegan un papel importante en la regulación del tono vascular, la permeabilidad vascular y la formación de nuevos vascular. disfunción de la célula endotelial está implicada en el desarrollo y la progresión de muchas enfermedades cardiovasculares que incluyen la enfermedad cardíaca isquémica. Para examinar la función y caracterización de células endoteliales coronarias, aislamiento de células es el primer paso y requiere alta pureza y cantidad para llevar a cabo los experimentos posteriores. Este protocolo describe un método eficaz para aislar las células endoteliales coronarias de ratón adulto. El corazón de ratón se diseca y picada en trozos pequeños. Después de la digestión del corazón usando dispasa y colagenasa II, las células se lavaron y se incubaron con perlas magnéticas que están conjugados con el anticuerpo anti-CD31. Las perlas con las células endoteliales son lavados varias veces y están listos para usar en diversas aplicaciones, incluyendo formación de imágenes y EXPERIME biológica molecularNTS. Aislamiento eficaz rinde aproximadamente 10 4 células por un solo corazón con más del 90% de pureza.

El éxito en el aislamiento de las células endoteliales coronarias de un corazón de ratón produce normalmente alrededor de 10 4 células con más del 90% de pureza con una forma de adoquines. Para una población pura de células endoteliales, se debe tener cuidado durante todo el protocolo para asegurar un ambiente estéril libre de cualquier contaminación. La aparición de células alargadas o células ampliadas dentro de la población indica contaminación con otros tipo...

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

Isolation of Mouse Coronary Endothelial Cells

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

Aislamiento de células endoteliales de ratón coronaria

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

isolation-of-mouse-coronary-endothelial-cells

Research

JoVE Journal

Biology

11.1K vistas.  University of Arizona, Tucson. 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.

Video: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

Aislamiento de células endoteliales endocardiales y coronarias de la pared libre ventricular del corazón de rata

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Investigación

Biología del Desarrollo

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 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 ...

Neurociencias

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

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

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

Aislamiento de microvascular endotelial Tubos de arterias de resistencia Ratón

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

Isolation of 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 Valve Endothelial Cells

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

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

Aislamiento de las células murinas de la válvula endoteliales

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

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

Isolation of 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 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. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Aislamiento de células endoteliales de la luz de arterias carótidas de ratón para experimentos multiómicos unicelulares

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...