This work was supported by The British Heart Foundation and The Oak Foundation.

Realice todos los experimentos con animales de acuerdo a los protocolos aprobados por el Comité Institucional de Uso y Cuidado de Animales de Laboratorio. 1. Preparación de Medios de Cultivo <ol><li> Hacer medios de cultivo para la línea celular de fibroblastos humanos CCL-153: F-12K mediana, suero bovino fetal al 10% (FBS) y 100 U / ml de penicilina y estreptomicina. </li><li> Hacer reprogramación de medios para la generación de PIPSC: medio de Eagle modificado de Knockout Dulbecco (DMEM) que contiene 20% suero sustitución Knockout, 0,1 mM β-mercaptoetanol, 0,1 mM Medio Esencial Mínimo (MEM) de ácidos no esencial Amino, 10 ng / factor de crecimiento de fibroblastos básico ml 2 (bFGF-2) y 100 U / ml de penicilina y estreptomicina. Añadir bFGF-2 frescos cada vez antes de usar los medios de comunicación. </li><li> Hacer Diferenciación Media (DM) para inducir células del músculo liso (SMC) Diferenciación: α-MEM medios que contienen 10% de FBS, Factor de Crecimiento 0,1 mM β-mercaptoetanol, 100 U / penicilina y estreptomicina y 25 ml ng / ml derivado de las plaquetas(PDGF-ββ). </li><li> Hacer endotelial Diferenciación Media (EC-DM) para inducir células endoteliales (CE) Diferenciación: medios que contienen 50 ng de crecimiento endotelial medios de comunicación-2 (EGM-2) / ml factor de crecimiento vascular endotelial (VEGF) y 100 U / ml de penicilina y estreptomicina. </li></ol> 2. Reprogramación fibroblastos humanos en células madre pluripotentes inducidas (parcialmente PIPSC) <ol><li> Cultura línea celular de fibroblastos humanos CCL-153 en 0.04% de la solución de gelatina frascos revestidos en medio de cultivo. </li><li> Células Passage cada 3 días en una proporción de 1: 6. Utilice las células antes del paso 9 para una óptima eficiencia de la generación PIPSC. Las células están listas para la transfección al llegar a 80 - 90% de confluencia. </li><li> Linealizar pCAG2LMKOSimO plásmido policistrónico que contiene 4 factores de reprogramación octamer vinculante factor de transcripción 4 (OCT4), Sox2, factor de Kruppel-like 4 (KLF4) y C-MYC (pCAG-OSKM). Digerir 5 g de plásmido con 5 unidades de enzima de restricción PvuI durante 3 horas a 37 ° C. Purificarel plásmido con un kit comercial según el protocolo del fabricante. </li><li> Transfectar 2 x 10 6 fibroblastos humanos con 4 g de plásmido pCAG-OSKM linealizado mediante electroporación con el kit de fibroblastos dérmicos humanos (NHDF) nucleofection según el protocolo del fabricante. Sembrar suavemente las células transfectadas en pre 0,04% de gelatina recubierta matraz T25 que contiene 5 ml de pre-calentado reprogramación de medios. </li><li> Después de 24 horas, cambio de medio con suplementos de 25 mg / ml de neomicina para seleccionar una población pura de células transfectadas. </li><li> Cambio de medio cada dos días hasta el día 4. Nota: Los fibroblastos humanos se convierten en PIPSC después de 4 días de reprogramación. </li></ol> 3. Diferenciación de PIPSC en células endoteliales y musculares lisas <ol><li> PIPSC Seed en platos de colágeno IV pre-recubiertos que contienen DM durante 4 días para inducir la diferenciación en las SMC. Cambio de medio cada dos días hasta el día 4. </li><li> PIPSC Semilla de colágeno IV pre-recubierto platos containing endotelial-DM durante 6 días para inducir la diferenciación en las células endoteliales. Cambie medio cada dos días hasta el día 6. </li></ol> 4. Descelularizado Aorta Preparación del injerto Nota: Todas las soluciones y equipos deben ser estériles. <ol><li> Sacrificar el ratón por dislocación cervical y fijar el ratón en una posición supina bajo microscopio de disección. </li><li> Corte a través del esternón y alrededor de la caja torácica para abrir la cavidad torácica. Quite el corazón, los pulmones y el esófago para exponer la aorta. Retire con cuidado la grasa peri-aorta con fórceps. </li><li> Cortar la aorta desde el extremo anterior con unas tijeras y coloque un lado del extremo con fórceps romos. Separe la aorta de la columna de la columna vertebral por detrás mediante disección roma. Cierre con cuidado todas las arterias de ramificación de la aorta por la ligadura con un electrocoagulador bipolar y el corte desde el otro extremo de la ligadura con unas tijeras. </li><li> Utilice una jeringa de 5 ml para limpiar el lumen de la aorta con 3 ml de solución salina que contiene 100 U de heparina para prevenir la formación de coágulos de sangre. </li><li> Cortar el extremo posterior de la aorta antes se ramifica en el renal. Mantener la integridad de la aorta es muy importante durante todo el procedimiento. </li><li> Enjuague el lumen de la aorta con 5 ml de dodecilsulfato de sodio (SDS) solución de 0,075% diluido en tampón fosfato salino (PBS). </li><li> Remojar la aorta torácica en solución de SDS al 0,075% en una placa de Petri de 10 cm. Coloque la placa de Petri en un agitador orbital durante 2 horas a 150 rpm a temperatura ambiente. </li><li> Enjuague el lumen de la aorta con 5 ml de PBS. </li><li> Remoje la aorta en PBS en una placa de 10 cm de Petri. Coloque la placa de Petri en el agitador orbital durante 2 horas a 150 rpm a temperatura ambiente. Actualizar PBS cada 20 minutos. </li><li> Enjuague el lumen de la aorta con 5 ml de PBS. </li><li> Mantenga injerto aorta descelularizado en PBS a 4 ° C durante un máximo de una semana. </li></ol> 5. Dual Siembra de PIPSC y biorreactor acondicionado Nota: Todas solución des y el equipo deben ser estériles. Realizar todas las operaciones en una campana de cultivo de tejidos. <ol><li> Montar el circuito de flujo de biorreactor, como se ilustra en la Figura 1. Conectar cámara de incubación, depósito de medio, la bomba peristáltica y la cámara de cumplimiento de la tubería. Volumen mínimo de medio necesario para la circulación es de 40 ml. </li><li> Pre-condición del recipiente descelularizado con medio de cultivo empapando la aorta en un medio de DM en un plato de 10 cm Petri. Coloque la placa de Petri en el agitador orbital durante 1 hora a 50 rpm a temperatura ambiente. </li><li> Introduzca 1 cm tubos de nylon de longitud (OD 0,9 mm, ID 0,75 mm) en ambos extremos de la vasija descelularizado bajo el microscopio. Ate el recipiente y los tubos con 8-0 suturas de seda. </li><li> Montar el injerto de aorta descelularizado en la cámara de incubación mediante la conexión de los tubos de nylon con los puertos de entrada y salida (1/32 &quot;) de tubos de la pared de la cámara. Mantener cámara de incubación con 5 ml de medio cada vez. </li><li> Trypsinize PIPSC añadiendo precalentadotripsina para cubrir la placa de cultivo y el rock suavemente el plato 10 veces. Deseche la tripsina y dejar el plato en la incubadora durante 2 - 3 minutos. Añadir medio de cultivo pre-calentado en el plato y mezclar el medio con las células. </li><li> Contar el número de células usando un hemocitómetro. Centrifugar dos alícuotas de suspensiones celulares que contienen 5 x 10 5 células cada uno durante 5 min a 300 x g. Aspirar el sobrenadante por completo. </li><li> Después de aspirar el sobrenadante por completo, resuspender 1 pellet de las pepitas sedimentos celulares en 50 l de DM. Inyectar esta suspensión de células en la luz del vaso descelularizados. </li><li> A continuación, volver a suspender el sedimento celular otra pips en 100 l de Matrigel. Pipetear con cuidado la mezcla sobre el injerto de aorta descelularizado. </li><li> Espere de 10 - 15 min hasta que la mezcla se convierte en un estado similar a un gel uniformemente y se envuelve alrededor de la superficie externa del vaso. Llene la cámara de incubación con DM. </li><li> Configuración de lugar en una incubadora de CO2 al 5% todo el biorreactor a 37 ° C. Girar manualmente el injerto descelularizado 90 ° alrededor del eje longitudinal cada media hora en el primero 2 hr. </li><li> Mantenga la cultura estática durante 12 horas para permitir la adhesión celular. </li><li> Entregar DM a través del lumen por la bomba peristáltica para inducir la diferenciación de células musculares lisas. Iniciar la velocidad de flujo inicial en 5 ml / min y aumento escalonado a 20 ml / min durante 24 hr. </li><li> Mantener la tasa de flujo del medio circulante a 20 ml / min durante 24 horas. </li><li> Detener el flujo del medio y mover el ajuste de la incubadora biorreactor. </li><li> Re-semilla del lumen del injerto con PIPSC. Trypsinize, recuento y centrifugar 1 x 10 6 PIPSC como en los pasos 5.5 a 5.6. Resuspender las células en 50 l de medio EGM-2 que contienen 50 ng / ml de VEGF. Inyectar la mezcla de células en el lumen del injerto. </li><li> Cambiar el medio en la cámara de incubación y toda la circulación en EGM-2 medio que contiene 50 ng de VEGF / ml para inducir la diferenciación endotelial. </li><li> Mueva retrasando a th el biorreactore incubadora. Gire manualmente el injerto 90 ° cada 30 minutos en las primeras 2 horas y luego mantener una cultura estática durante 12 horas. </li><li> Iniciar la circulación de flujo de 5 ml / min y aumento escalonado a 35 ml / min. Mantener el caudal a 35 ml / min durante 5 días. Cambiar circulante y medio cámara de cada dos días. </li><li> Se recoge el injerto de ingeniería para su posterior injerto de ratón. </li></ol> 6. El injerto de doble cabeza de serie PIPSC injerto en ratones <ol><li> Uso NOD.CB17-Prkdc scid / NcrCrl ratones macho como receptores de injertos buque. Asegúrese siempre de que los ratones son alrededor de 10 semanas de edad, pesan aproximadamente 25 - 35 g, y se encuentran en buenas condiciones de salud. </li><li> Anestesie ratón receptor mediante la administración de una combinación de Hypnorm (25 mg / kg) y Hypnovel (25 mg / kg) por vía intraperitoneal. Aplicar vaselina pomada oftálmica en los ojos para evitar la sequedad mientras que bajo anestesia. Confirme anestesia eficiente, garantizando que los ratones tienen los músculos relajados y están respirando constantemente. <ol><li> Fijar el ratón en una posición supina con su cuello afeitó y extendido. Administrar sulfato de atropina (1,7 mg / kg) en combinación con los anestésicos para asegurar que las vías respiratorias del ratón sigue siendo clara. </li></ol></li><li> Preparar una incisión en la línea media de la mandíbula al esternón del ratón. Bajo un microscopio de disección con 5 a 10 veces la amplificación, levantar las glándulas salivales derecha lateralmente y eliminar el músculo esternocleido- derecho a exponer la arteria carótida común derecha. </li><li> Retire con cuidado los tejidos unidos a movilizar a la arteria carótida común derecha desde el extremo distal hacia el extremo proximal. Ligar la porción media de la arteria carótida común dos veces con una sutura de seda 8-0 y disecar entre los dos lazos. </li><li> Pasar a través de un manguito de tubo de nylon autoclavable en cada extremo del vaso y fijar cada extremo con pinzas microhemostat. </li><li> Quitar el lazo de sutura en un extremo y aplicar una gota de heparina (100 U / ml). Inmediatamente después, invertir el extremo distal del ésimoe arteria para cubrir todo el cuerpo del manguito y fijar el recipiente invertido con una sutura de seda 8-0 en el manguito. <ol><li> Repetir el mismo procedimiento para la otra porción de la arteria. Arteria Lavar con solución salina para eliminar los coágulos de sangre. </li></ol></li><li> Implantar un injerto de vaso descelularizado entre dos extremos de la arteria carótida por deslizamiento los extremos de los vasos de la descelularizados a través de los puños de la arteria y la fijación con suturas de seda 8-0. Eliminar pinzas vasculares de cualquiera de los extremos antes de evaluar la pulsación del injerto. </li><li> Coloque la glándula salival de vuelta a su posición original. Cierre de la piel en la ubicación quirúrgica con una sutura interrumpida usando una sutura de poliglactina 6-0. </li><li> Consistentemente observar los signos vitales de ratón después de que se completa el procedimiento, hasta que se reanude la conciencia. Sacrificar los ratones receptores por dislocación y de los vasos de la cosecha injertos cervicales ya sea 24 horas o 3 semanas más tarde para su posterior análisis. </li></ol>

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Los autores no tienen conflictos de intereses financieros.

El protocolo actual indica un sonido, la estrategia rápido, simple, eficiente y reproducible en la que los vasos de ingeniería tisular funcionales se pueden generar utilizando PIPSC a partir de fibroblastos humanos. Esta técnica representa una herramienta valiosa para la medicina regenerativa, la ingeniería de tejidos y terapia celular específica del paciente potencialmente en el futuro cercano. Los pasos críticos para garantizar la eficacia del protocolo incluyen la preparación de PIPSC, preparación de injertos...

La construcción de conductos vasculares es una estrategia fundamental para la reparación quirúrgica de los vasos dañados y heridos resultantes de enfermedades cardiovasculares. Hasta la fecha, los materiales utilizados en la cirugía de injerto biocompatible incluyen polímeros sintéticos (politetrafluoroetileno [teflón], politetrafluoroetileno expandido [ePTFE; Gore-Tex] o tereftalato de polietileno [Dacron]), aloinjertos, tejido autólogo (pericardio o vena safena) y xenoinjertos 1. Mientras que los injertos artificiales (por ejemplo, Gore-Tex y Dacron) son los más utilizados, estos materiales probablemente causan numerosas complicaciones a corto y largo plazo que incluyen la estenosis, la deposición de calcio, trombo-embolia y las infecciones. Aunque los pacientes con injertos biológicos presentes con una disminución de eventos tromboembólicos, todavía se encuentran con limitaciones, como el fracaso del injerto secundaria y durabilidad acortada debido a la degradación de la calcificación 2. Por lo tanto, a pesar de las mejoras significativas en t quirúrgicaechniques largo de los años, los investigadores y los médicos todavía están cargados con la necesidad de identificar el conducto ideal para enfermedades vasculares. Más recientemente, el campo de la investigación de la ingeniería de tejido vascular ha generado un concepto en el que las células se incorporan en estructuras biodegradables, con el objetivo de crear un entorno biomimético que personifica un recipiente funcional para injerto realizado con éxito 1. Fundamentalmente, el éxito de las construcciones vasculares depende de tres componentes esenciales; las células que componen el andamio, es decir, una capa interna de células endoteliales y una capa de células del músculo liso, un andamio que contiene la matriz extracelular apropiado para proporcionar propiedades mecánicas comparables a la vasculatura nativa, y la señalización molecular / celular que se requiere para iniciar / regulador reparación. La permeabilidad del injerto a largo plazo y el desarrollo sostenido de los neo-tejidos son altamente dependientes de la siembra de células eficaz de andamios, THereby haciendo que la decisión del tipo de célula de importancia crítica. Varios informes demuestran el uso de endoteliales maduras y las células musculares lisas de diferentes fuentes para desarrollar conductos de pequeño diámetro de 3-6. Aunque prometedores, la falta de vasos autólogos suficiente para obtener endoteliales maduras y las células musculares lisas siguen siendo una carga considerable. Más recientemente, las células madre a partir de diversas fuentes han sido explotados para aplicaciones de ingeniería de tejidos vasculares. Células mononucleares de hecho, las células madre pluripotentes una variedad de tipos de células madre, incluyendo células madre embrionarias 7, inducida (CMPI) 8,9, PIPSC 10,11, derivadas de la médula ósea 12, células madre mesenquimales 13, las células progenitoras endoteliales y la pared del vaso adulto -derivado células madre antígeno-1 (Sca-1) + células madre / progenitoras 14,15 todos se han demostrado que son capaces de diferenciarse en células endoteliales ya sea funcional o de músculo liso en respuesta a los medios de comunicación definidos ycondiciones de cultivo. Además, la capacidad ilimitada de auto renovación de las células madre a tomar mejores candidatos a diferencia endoteliales maduras y las células musculares lisas que sólo se puede dividir por un número finito de veces antes de someterse a la detención del crecimiento y la senescencia. La selección del material de andamio para generar tejido éxito recipiente diseñado para el injerto depende de varios factores tales como la biocompatibilidad, propiedades biomecánicas, y la velocidad de biodegradación. Fundamentalmente, los materiales utilizados para crear andamios para los injertos deben ser biodegradables y no se montarán las respuestas inmunes receptores innecesario. Además, debe abarcar una porosidad adecuada y la microestructura para la fijación celular y la supervivencia posterior. Hasta la fecha, los materiales más comunes usados ​​para andamios en la ingeniería de tejidos vascular incluyen polímeros de ácido poliglicólico, ácido poliláctico, y poli ε-caprolactona 16. Más recientemente, los materiales biológicos descelularizados tienenTambién ha aplicado con cierto éxito. Varios laboratorios han demostrado que la siembra de los vasos porcina humana descelularizado, canino o con células autólogas proporciona un injerto biológico que resistió la coagulación y la hiperplasia intimal 17-19. Otras estrategias de ingeniería de tejidos vascular incluyen injertos vasculares extracelulares basado proteínas de matriz por ejemplo, células de siembra en gel de fibrina 13 y generando láminas de células sin el apoyo del andamio 20, 21. El protocolo actual demuestra la diferenciación de PIPSC humano en endotelial funcional y células musculares lisas, la generación de un biorreactor que consiste en un andamio recipiente descelularizado para albergar células vasculares derivadas de PIPSC funcionales, y el injerto de los vasos de ingeniería tisular en la inmunodeficiencia combinada severa (SCID ) ratones. PIPSC son un tipo de célula óptima a utilizar para la ingeniería de tejidos de injertos de vasos ya que estas células no forman tumores en ratones o plantean ético yallo respuestas inmunes. Por otra parte, hemos demostrado que la estrategia para la generación de células musculares Pipa-células endoteliales y Pipa-lisas es eficiente y reproducible 10,11. A partir de entonces, se diseñó un recipiente de descelularizado para la siembra de las células vasculares derivadas de PIPSC para imitar las proteínas de la matriz que existe dentro de un vaso nativo, mejorando así la supervivencia de injerto y la eficacia. Además, la descelularización de los vasos antes de la siembra PIPSC previene la aparición de las respuestas inflamatorias montadas por tipos de células inmunes, tales como macrófagos. Más importante aún, este protocolo no sólo representan una metodología para generar prometiendo conductos vasculares para la traducción a los seres humanos, sino que también proporciona medios valiosos para el estudio y la comprensión de los mecanismos moleculares que regulan la regeneración del tejido vascular a través de modelos de ratón.

<table><tbody><tr><td>Human Fibroblasts CCL-153</td><td>ATCC</td><td>CCL-153</td><td>Prenatal human embryonic fibroblasts</td></tr><tr><td>ATCC F-12K Medium (Kaighn&#039;s Modification of Ham&#039;s F-12 Medium)</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Knockout DMEM medium optimized for embryonic stem cells</td><td>Life technologies (Gibco)</td><td>12660-012</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life technologies (Invitrogen)</td><td>10828-028</td><td></td></tr><tr><td>Human Basic FGF-2&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-837</td><td></td></tr><tr><td>alpha-MEM medium</td><td>Life technologies (Invitrogen)</td><td>32571093</td><td></td></tr><tr><td>Human PDGF</td><td>R&amp;amp;D System</td><td>120-HD-001</td><td></td></tr><tr><td>Gelatin Solution 2%</td><td>Sigma</td><td>G1393</td><td></td></tr><tr><td>Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td><td>Addgene</td><td>20866</td><td></td></tr><tr><td>&amp;nbsp;PvuI Restriction Enzyme</td><td>New England Biolabs</td><td>RO150S</td><td></td></tr><tr><td>SureClean Plus</td><td>Bioline</td><td>BIO-37047</td><td></td></tr><tr><td>Nucelofection Kit (NHDF Kit)</td><td>LONZA</td><td>VPD-1001</td><td></td></tr><tr><td>Neomycin</td><td>SIGMA</td><td>G418</td><td></td></tr><tr><td>KL 1500 LCD, Illumination for Stereo Microscopy</td><td>SCHOTT</td><td>KL 1500 LCD</td><td>Cold light illumination for stereo microscopy</td></tr><tr><td>Nikon Zoom Steromicroscope SMZ800</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>10% SDS Stock Solution Molecular Biology Reagent</td><td>Severn Biotech</td><td>CAS 151-21-3</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Matrigel (10mg/ml)</td><td>BD</td><td>A6661</td><td></td></tr><tr><td>Shaker IKA Vibrax with Shaking platform VX 7&amp;nbsp;</td><td>Jepson Bolton&#039;s, Janke&amp;amp;Kunkel</td><td>S32-102</td><td></td></tr><tr><td>Masterflex L/S Digital Pump Drive</td><td>Cole-Parmer</td><td>WZ-07523-80</td><td></td></tr><tr><td>Masterflex L/S 6-channel, 6-roller cartridge pump head</td><td>Cole-Parmer</td><td>EW-07519-15</td><td></td></tr><tr><td>Masterflex L/S large cartridges for pump head</td><td>Cole-Parmer</td><td>EW-07519-75</td><td></td></tr><tr><td>Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td><td>Cole-Parmer&amp;nbsp;</td><td>WZ-96410-14</td><td>Tubing goes through the peristaltic pump</td></tr><tr><td>0.5mm ID, 0.8 mm OD Silicone Tubing</td><td>SILEX</td><td>N/A</td><td>Tubings connect incubation chamber, media reservoir and compliance chamber&amp;nbsp;</td></tr><tr><td>Fitting Reducer 0.5 to 1.6, natural Polypropyline</td><td>Ibidi</td><td>10829</td><td>Adapter connect above two types of tubings</td></tr><tr><td>1/32&quot; Tubing, ID 0.01&quot; (250&amp;micro;m) Material: PEEK</td><td>LabSmith</td><td>T-132-010P</td><td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td></tr><tr><td>One-Piece Fittings&amp;nbsp;</td><td>LabSmith</td><td>T-132-100</td><td>Fix the above tubings through the incubation chamber wall</td></tr><tr><td>Nylon tubes (OD 0.9mm, ID 0.75mm)&amp;nbsp;</td><td>Smiths Medical</td><td>N/A</td><td>Tubings insert into two ends of the aorta graft</td></tr><tr><td>NOD.CB17-Prkdc&lt;sup&gt;scid&lt;/sup&gt;/NcrCrl mouse</td><td>Charles River</td><td></td><td></td></tr><tr><td>Surgical sutures, 8-0&amp;nbsp; silk</td><td>ETHICON</td><td>W819</td><td></td></tr><tr><td>Hypnorm</td><td>Vetapharm</td><td>Vm21757/4000</td><td>Neuroleptanalgesic for use in mice</td></tr><tr><td>Hypnovel (Midazolam)</td><td>Roche</td><td>59467-70-8</td><td>Induction of anaesthesia</td></tr><tr><td>Dissecting microscope</td><td>Carl Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Nylon Tubing</td><td>Portex LTD</td><td>800/200/100/200</td><td>0.65 mm in diameter and 1 mm in length; to make artery cuff</td></tr><tr><td>Electrocoagulator</td><td>Martin&amp;nbsp;</td><td>SN 54.131</td><td>Ligation of artery branches on aorta</td></tr><tr><td>Bipolar micro hemostat forceps</td><td>Martin</td><td>80-91-12-04</td><td>Fixation of vessel ends</td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFX-7</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>JFL-3dZ</td><td></td></tr><tr><td>Vessel Dilator</td><td>S&amp;amp;T</td><td>D-5aZ</td><td></td></tr><tr><td>Mini applier&amp;nbsp;</td><td>AESCULAP</td><td>FE572K</td><td></td></tr><tr><td>Micro hemostats clips</td><td>AESCULAP</td><td>FE720K</td><td></td></tr><tr><td>Surgical sutures, 6-0 VICRYL</td><td>ETHICON</td><td>V489</td><td></td></tr></tbody></table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

La generación exitosa de PIPSC fue confirmado 4 días después nucleofecting fibroblastos humanos con un plásmido linealizado pCAG2LMKOSimO llevar 4 factores de transcripción, Oct4, SOX2, KLF4 y c-myc (OSKM). PIPSC muestra un fenotipo notablemente distinta en comparación con fibroblastos (Figura 2A) y expresó los 4 factores de reprogramación en el mRNA (Figura 2B) y los niveles de proteína (Figura 2C) 10. La eficacia de un injerto vascular a base de PI...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

A continuación, presentamos un protocolo para generar ingeniería tisular injertos de vasos que son funcionales para el injerto en ratones por doble siembra de células madre pluripotentes inducidas parcialmente (PIPSC) - deriva células musculares lisas y PIPSC - células endoteliales derivadas de un biorreactor andamio buque descelularizado.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.9K vistas.  King's College London BHF Centre.  A continuación, presentamos un protocolo para generar ingeniería tisular injertos de vasos que son funcionales para el injerto en ratones por doble siembra de células madre pluripotentes inducidas parcialmente (PIPSC) - deriva células musculares lisas y PIPSC - células endoteliales derivadas de un biorreactor andamio buque descelularizado. 

Video: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

El trasplante ortotópico aórtica en los ratones para el Estudio de la Enfermedad Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Investigación

Medicina

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Modelos de ratón para Arteriosclerosis Graft

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Técnica quirúrgica para la implantación de la ingeniería tisular injertos vasculares y posterior<em&gt; En Vivo</em&gt; Monitoreo

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Bioingeniería

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino Cervical Trasplante Cardiaco Modelo Usando una técnica de Cuff Modificado

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Implantación de la vena cava inferior interposición del injerto en modelo de ratón

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

El escalado de Engineered injertos vasculares Usando 3D Impreso guías y el método de anillo de apilamiento

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Un microquirúrgica Modelo Novela de heterotópico, en bloque, pared torácica, el timo y el trasplante cardiaco en ratones

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Siembra e implantación de un injerto traqueal tejido-dirigida biosintético en un modelo murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Utilizando la técnica de la manga en un modelo murino de trasplante aórtico - un Video instructivo

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

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