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><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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&amp;nbsp;</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 &amp;deg;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&#039;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&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;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&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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 %&amp;nbsp;</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)&amp;nbsp;</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)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;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 &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</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

Universidad de Cartagena UDEC

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 of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Aislamiento de células endoteliales humanas de Colon Normal y el Carcinoma colorrectal - un protocolo mejorado

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Investigación

Investigación sobre el cáncer

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Aislamiento de células endoteliales progenitoras de la sangre de cordón Umbilical humano

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Biología del Desarrollo

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Aislamiento de células tumorales de colon humanos primarios de tejidos quirúrgicos y cultivándolas Directamente en Elastic sustratos blandos de tracción Citometría

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Bioingeniería

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Biología

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Neurociencias

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Un modelo de xenoinjerto derivado del paciente para la malformación venosa

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

En Enriquecimiento vitro de células iniciadoras del tumor del cáncer de ovario

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Medicina

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 ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

El aislamiento de células madre CD133 + hígado para la expansión clonal

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.