ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

El siguiente protocolo se lleva a cabo de acuerdo con las directrices establecidas por el Departamento de Medicina de Laboratorio Animal de la Universidad de Carolina del Norte en Chapel Hill. 1. Prepare el siguiente material y reactivos Antes de empezar <ol><li> Preparar los medios de comunicación CE completándola 400 ml bajo nivel de glucosa (1 g / L D-glucosa o LG) de Dulbecco Modificado medio de Eagle (DMEM) con 50 ml de suero bovino fetal inactivado por calor, 50 ml Nu-Serum IV, 5 ml antibiótico-antimicótico, y el hFGF, VEGF, hEGF, R3-IGF-1, y los componentes de heparina desde el kit comercial. </li><li> Preparar 500 ml de tampón FACS (0,5% de BSA y EDTA 2 mM en PBS); filtrado a través de un 0,22 micras taza del filtro estéril. </li><li> Esterilizar o desinfectar el tablero de la disección. </li><li> Esterilizar pasadores de disección, tijeras quirúrgicas, y disectores. </li></ol> 2. Aislamiento CE (Día 1, ~ 5 horas) <ol><li> La eutanasia ratón con el dióxido de carbono u otros métodos ingenio compatibleel Comité de Cuidado y Uso de Animales Institucional (IACUC) políticas h. Nota: Múltiples tumores mamarios varían en tamaño (5 - 15 mm de diámetro) pueden desarrollar en un solo ratón modificado genéticamente como C3-TAG, asegúrese de recoger todos ellos para el aislamiento TEC. Si el uso de los tumores que se ortotópicamente injertan en los ratones, piscina de dos a tres 1 cm 3 tumores para un solo aislamiento CE. Aquí usamos tumores mamarios como una demostración, pero el protocolo se puede modificar para otros tipos de tumores. </li><li> Desinfectar ratón por pulverización o limpiando el lado ventral del ratón con una amplia cantidad de 75% v / v de etanol. </li><li> Resecar tumores con un par de tijeras y disectores utilizando técnicas asépticas en una campana estéril o en un banco limpio. <ol><li> Extiende y el pin de las extremidades de los ratones en una tabla de disección. Haga una incisión en la línea media ventral con las tijeras sin necesidad de abrir el peritoneo. Diseccionar lateralmente entre la piel y el peritoneo hacia las glándulas mamarias donde se encuentran los tumores.No abra el peritoneo. </li><li> Impuestos Especiales sólo el tejido tumoral de las glándulas mamarias, dejando de lado los márgenes mamarias normales. Recortar con cuidado los tejidos no tumorales, como la piel y los músculos, y coloque los tumores disecados en un tubo cónico que contiene 30 ml de LG-DMEM en hielo. </li></ol></li><li> Traiga muestras tumorales a campana de cultivo de tejidos; lavar tejidos con estéril LG-DMEM 1 - 2 veces. </li><li> Tumores de transferencia del tubo cónico a un cultivo de tejidos placa de Petri estéril, añadir 2 ml de DMEM-LG en el plato, y picar con un par de tijeras estériles en pedazos &lt;5 mm. </li><li> Añadir 5 ml de colagenasa (stock = 2 mg / ml en solución salina equilibrada de Hank, en lo sucesivo HBSS), 1 ml de dispasa (stock = 2,5 U / ml en HBSS) y 75 l de desoxirribonucleasa (stock = 1 mg / ml en PBS ) en la placa de Petri. Volumen total es ahora ~ 10 ml. </li><li> Transfiera la mezcla colagenasa / tejido de la placa de Petri a un tubo de tejido-disociador y ejecutar en un disociador de tejido durante 60 segundos dos veces (pre-establecido disociación prOGRAMA en el disociador: 1.270 rondas totales por corrida). Incubar con la luz agitando en un agitador durante 75 minutos a 37 ° C. </li><li> Filtrar digiere el tejido a través de un filtro de células de 100 micras sobre un tubo cónico de 50 ml. Filtro Enjuague con 5 ml de tampón FACS para lavar las células restantes. Giran a 280 xg durante 5 min y aspirar cuidadosamente el sobrenadante sin alterar el sedimento celular. </li><li> Diluir 1 ml de tampón de lisis RBC de stock (10x) en 9 ml de agua estéril. Lisar células rojas de la sangre con 10 ml de tampón de lisis (1x), e inmediatamente giran 5 min a 280 x g. Nota: Este paso se puede omitir si poco de sangre es visible. </li><li> Resuspender en 10 ml de tampón FACS. Mezclar 10 l de suspensión celular con 10 l de azul de tripano, y contar las células vivas utilizando un hemocitómetro. </li><li> Resuspender las células en ~ 10 7 células / 100 l. Añadir 10 l de bloque de FcR por 100 l de suspensión celular, y se incuba en hielo durante 10 min. </li><li> Añadir el anticuerpo CD31 de rata anti-ratón conjugado con PE según. a la Tabla 1 incubar en hielo durante 10 - 15 min y el tubo de película de vez en cuando. </li><li> Añadir 10 ml de tampón FACS para el tubo y centrifugado a 280 xg durante 5 min. Retirar con cuidado el sobrenadante, y se lava el sedimento de células de nuevo con 5 ml de tampón FACS. Centrifugar para sedimentar las células. Aspirar el sobrenadante sin sedimento celular perturbador. </li><li> Añadir tampón y anti-PE FACS microperlas de acuerdo con la Tabla 2 Incubar en hielo durante 10 -. 15 min. Tubo de Flick de vez en cuando. </li><li> Añadir 10 ml de muestras de tampón FACS y centrifugado a 280 xg durante 5 min; lavar una vez con 5 ml de tampón FACS y se centrifuga de nuevo. Aspirar el sobrenadante sin perturbar pellet. </li><li> Traiga volumen a 300 l de tampón FACS. Gira a 35 micras de células colador tapado tubo 5 min a 280 x g. Utilice 2 tubos y un volumen mayor si es necesario. </li><li> Establecer Multistand magnético y columnas magnéticas en el capó, adjunte una columna para el separador y equilibrar la columna con 2 ml de tampón FACS. </li><li> Aspirate el sobrenadante y resuspender el sedimento celular en 0,5 - 1 ml de tampón FACS. </li><li> Pasar la suspensión celular a través de la columna magnética equilibrada. </li><li> Lavar la columna tres veces con 2 ml de tampón FACS, y recoger flujo a través (FT) en un tubo de 15 ml (fracción FT). </li><li> Tomar la columna fuera del separador y se eluye con 2 ml de tampón de FACS en otro tubo de 15 ml (fracción de eluato). Utilice émbolo para asegurar que todas las células están fuera de la columna. Repita la elución dos veces más con 2 ml de tampón FACS cada vez. </li><li> Haga girar el eluido a 280 xg durante 5 min. </li><li> Eliminar el sobrenadante y resuspender el precipitado en 10 ml de los medios de comunicación de la CE. </li><li> Igualmente dividir la fracción de eluato (~ 6 ml) en platos recubiertos de gelatina 10 cm. Durante tres 1 cm 3 tumores, placa de células eluye en al menos cuatro placas. Células Alternativamente, la placa eluyó a diferentes concentraciones en varias placas (por ejemplo., Sembrar 0,5 ml, 1 ml, 1,5 ml y 3 ml de eluido en cuatro placas) para asegurar que unt menos una placa está escasamente siembra con células eluidas. Compruebe que la confluencia de las células unidas se encuentra en ~ 1% al día siguiente (es decir, aproximadamente 1,0 - 2,0 x 10 5 células unidas). Nota: Las células necesitan ser chapado escasa de modo que las colonias de la CE pueden formar sin estar contaminados por otros tipos de células. </li><li> Fracción FT placa en una placa de 10 cm para permitir células recuperan O / N. Compruebe que la placa es de 80 - 100% de confluentes al día siguiente. Congele por las células (-80 ° C) en 250 medios de congelación de células mu l en un criotubo y almacenarlas en nitrógeno líquido al día siguiente. Nota: fracción FT se puede utilizar para el aislamiento de células tumorales en una etapa posterior y / o como un control negativo para el análisis de la expresión génica CE de los clones aislados de la CE. </li><li> Cambie los medios de comunicación cada 2 - 3 días. Las colonias empiezan a formar después de 7 - 10 días. Colonias pequeñas de la CE pueden ser identificados ya en el día 3. Marque las colonias con un marcador de punta fina en el fondo del plato. </li><li> Raspe c inespecíficaells rodean las colonias identificadas con una punta pippet 200 l estéril. </li></ol> 3. Colonia Selección Anillos Usando Clonación <ol><li> Cambie los medios de comunicación cada 2 - 3 días. Pureza CE se puede comprobar por las LDL (Dil-Ac-LDL) Además en unos 5-7 días. Añadir 50 l de LDL por 10 ml de medio CE y se incuba durante 3-4 horas antes de comprobar las células bajo el microscopio de fluorescencia. Nota: LDL Múltiple + clones de la CE se pudieron observar en ~ 7 días. </li><li> Comience cosechar colonias CE cuando alcanzan diámetros de 3 - 5 mm de tamaño. (Elija grandes colonias que están llenas de pequeñas de LDL + células para obtener mejores resultados.) </li><li> Antes de la cosecha de la CE con los anillos de clonación, pre-capa de unos 6 y placas con 0,5% de gelatina. Aspirar gelatina, añadir 2 ml de medio CE a cada pocillo, y mantener las placas en una incubadora hasta que se necesite. </li><li> Raspe no comunitario en los bordes de las colonias para asegurarse de que no hay otros tipos de células serán atrapados dentro del anillo de la clonación. </li><li> Usando unmicroscopio de contraste de fase (4X o 10X objetivo), se rodeará con un marcador de punta fina en la parte inferior de la placa de cultivo de las áreas que contienen colonias de la CE. </li><li> Lavar la placa con 10 ml de PBS y dejar una capa muy delgada de PBS en la placa cuando la aspiración. (Importante: una pequeña cantidad (~ 0,5 ml) de PBS mantendrá células vivas durante el procedimiento de anillo clonación; también, adhesivo tisular necesita agua para unir.) </li><li> Elija un anillo de clonación de tamaño apropiado. Use un par de pinzas de disección para recoger un anillo, y con una punta de pipeta de 10 l aplicar uniformemente una pequeña cantidad de adhesivo tisular en el anillo de la clonación. Nota: utilice sólo la cantidad mínima (~ 0,2 l de un pequeño anillo) de adhesivo tisular en los anillos de clonación, y crea adhesivo tisular seguro se extiende de manera uniforme alrededor de la superficie del fondo para asegurar un buen sellado. Adhesivo tisular excesivo produce calor y forma películas que pueden matar a las células. </li><li> Coloque el anillo de la clonación sobre la colonia CE. Presione suavemente el anillo de clonación para pegar los ring en el plato. Asegúrese de que las colonias no se secan antes de pegar el anillo. </li><li> Inmediatamente pipeta 25 l de solución de desprendimiento celular enzimática en el anillo de clonación e incubar ~ 1 min o hasta que las células están débilmente unidos. </li><li> Células de pipeta en el anillo de clonación gota a gota en un pocillo de una placa de 6 pocillos que contienen los medios de comunicación de la CE pre-calentado. (Importante: No agitar la placa para dispersar las células; CE prefieren crecer en racimos apretados.) Lave el anillo de la clonación con 50 - 100 medios de comunicación de la CE mu l recoger tantas células como sea posible, y la transferencia de todos los lavados en el mismo de 6 pocillos . </li><li> Si algunas colonias en la placa de 10 cm son demasiado pequeños para cosechar, añadir 10 ml de medio fresco, deje que las colonias crecen durante unos cuantos días, y repita el procedimiento de clonación anillo nuevo. </li><li> Crecer colonias cosechados en placas de 6 pocillos hasta 80-100% de confluencia, y la transferencia de células de 3 pocillos de una placa de 6 pocillos, antes de expandirse en una placa de 10 cm de cultivo de tejidos. Raspe células contaminantes. Repetirotra ronda de procedimiento anillo de clonación (Pasos de 03.05 a 03.10) si es necesario. Mantenga células relativamente confluente (~ 60-70%) cuando se expande. CE puede dejar de crecer si chapado demasiado escasos. </li><li> Caracterizar CE por FACS, tinción, PCR, etc. Tenga en cuenta que Dil-Ac-LDL es fluorescente y puede interferir con PE u otros anticuerpos fluorescentes para FACS. </li></ol>

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Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., 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=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, 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=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., 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+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</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 (2), 204-219 (2013).</li><li>Ingram, D. 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

Debido a las dificultades en la obtención de cultivos puros TEC primaria, muchos estudios in vitro TEC sustituto con las líneas de la CE disponibles comercialmente o CE primaria como la vena umbilical humana CE (HUVEC) 13. Sin embargo, estas poblaciones de la CE de los tejidos normales sólo se pueden servir como sustituto de la TEC que difieren notablemente de sus homólogos normales. Por ejemplo, TEC son fenotípicamente y funcionalmente anormal in vivo y algunas de estas anomalías pued...

Las células endoteliales (CE) son esenciales durante el desarrollo de tumores sólidos. A partir de la iniciación del cambio angiogénico en los tumores latentes a la difusión y la siembra de metástasis en sitios distantes, EC formar los conductos que suministran sangre, oxígeno y nutrientes para sostener el crecimiento del tumor 1. Como se ha sugerido recientemente, EC también tiene funciones de perfusión independiente y formar un nicho que soporta el crecimiento de células madre de cáncer y otras células del estroma tumoral 2-5. Por lo tanto, altamente purificado específico de tumor CE (TCE) para el cultivo in vitro permite estudios funcionales de rutina que arrojará luz sobre nuevos mecanismos moleculares que median la angiogénesis tumoral y la diafonía con células tumorales. CE son altamente especializados dependiendo del tejido de origen 6. Debido a la naturaleza heterogénea de diferentes tipos de tumores y el microambiente tumoral, TEC también puede mostrar características únicas que reflejan una especialización o específico de tumorf la vasculatura. Por ejemplo, no es sorprendente variabilidad en las firmas de expresión génica en TEC aisladas a partir de diferentes tipos o grados de tumores 7,8. Sin embargo, la co-purificación frecuente de no comunitario, especialmente los fibroblastos asociados al tumor y las células tumorales, con TEC puede confundir a los análisis de la expresión de todo el genoma. Estos tipos de células no deseadas son especialmente problemáticas en los estudios que se basan en a largo plazo en la expansión in vitro de cultivos de TEC. Aquí descrito es un método de alta fidelidad que produce consistentemente culturas CE puros de tumores y otros tejidos. Tras enriquecimiento columna inmunomagnética de las fracciones de la CE y la eliminación de co-purificado no comunitario, un paso anillo clonación adicional para capturar colonias CE puro se utiliza 9. Cada colonia se puede expandir en cultivo durante varios pasajes sin la aparición de contaminantes no comunitario. Este método también produce múltiples clones CE de un único procedimiento de aislamiento, que es ideal para el estudio de endoheterogeneidad telial. Además, se muestra que Cdh5 cre: / l ratones ZsGreen l / s reportero son una valiosa herramienta para la generación de la CE &quot;destino asignada&quot;, indelebles, que mantienen ZsGreen fluorescencia en la cultura 10. Con pequeños ajustes en el protocolo, este método debe ser adaptable a diferentes tipos de tumores o tejidos normales.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

Recién aisladas células endoteliales específicos de tumores (TEC) se pueden usar para explorar los mecanismos moleculares de la angiogénesis tumoral y servir como un modelo in vitro para el desarrollo de nuevos inhibidores de la angiogénesis para el cáncer. Sin embargo, a largo plazo en la expansión in vitro de células endoteliales murinas (CE) es un reto debido a la deriva fenotípica en la cultura (transición-endotelial-mesenquimal) y la contaminación con no comunitario. Esto es especialmente cierto para TEC que se outcompeted fácilmente por co-purificado fibroblastos o células tumorales en cultivo. Aquí, un método de aislamiento de alta fidelidad que se aprovecha de enriquecimiento inmunomagnética junto con la selección de la colonia y la expansión in vitro se describe. Este enfoque genera fracciones CE puros que son totalmente libre de contaminación de las células del estroma o tumorales. También se muestra que el linaje de trazado de Cdh5 cre: / l reportero ratones ZsGreen l / s, que se utiliza con el protocolo descrito en este documento, son una valiosa herramienta para verificar celularpureza como las colonias de la CE aislados de estos ratones muestran fluorescencia ZsGreen duradero y brillante en la cultura.

CE representan solamente una fracción pequeña de la población total de células en la mayoría de los tejidos adultos 11. Por tanto, es importante para digerir completamente el tejido recogido en una suspensión de una sola célula que asegura la liberación máxima de CE de la matriz extracelular (ECM) y los tejidos conectivos. En nuestra experiencia, la selección inmunomagnética CD31 mediada sólo proporciona fracciones enriquecidas de la CE, pero no puros; por lo tanto, un paso crucial es la eliminaci...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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

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

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

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

11.1K vistas.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Video: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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

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

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

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

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

Investigación

Medicina

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

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

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

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

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

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

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

El aislamiento, la cultura, y de imágenes de Human Fetal pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

Preparación y<em&gt; In Vitro</em&gt; Caracterización de Magnetized miR-modificado células endoteliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Evaluación de las propiedades anticoagulantes y antiinflamatorias de las células endoteliales mediante cultivo celular 3D y sangre no anticoagulada

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Los desequilibrios musculares: Pruebas y entrenamiento de fuerza funcional excéntrico isquiotibiales en poblaciones deportivas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...