Este trabajo está financiado por los Institutos Nacionales de Salud Grant R01 EB-008836 y HL083895 R01 (tanto a LEN). Podríamos agradecer a Daryl Smith, el soplador de vidrio de la Universidad, para la toma de los biorreactores de nuestra investigación.

Autoclave Ensamble y autoclave los tubos del sistema de caudal y componentes bio-reactor (biorreactor sí mismo y la tapa de silicona tapón) como se indica en la Figura 1 y Figura 2. Alimentación por sonda tiene un conector macho en un extremo y un extremo abierto en el otro lado. Tres segmentos de tubo corto se inserta a través de una gorra de silicona para el intercambio de gases. 1. Coser PGA Mesh <ol><li> Corte de malla de 1,1 cm PGA ~ x 8 cm hoja (depende del tamaño del biorreactor). </li><li> Limpie el tubo de silicona (diámetro interior de 3 mm) con agua destilada (dH20) y secar al aire antes de su uso. </li><li> Use Dexon 6.0 de sutura para coser PGA de malla alrededor del tubo de silicona limpia a partir de tres nudos quirúrgica seguida de puntos individuales. </li></ol> 2. PGA andamios Tratamiento de Superficies <ol><li> Sumerja los andamios de la PGA en NaOH 1 M de 1-2min y registrar el tiempo de tratamiento y utilizar el mismo tiempo para todos los andamios de la PGA. </li><li> Enjuague los andamios de la PGA en baños dH2O durante 2 minutos, 3 veces. </li><li> Pat PGA andamios seco con Kimwipes entre cada inmersión en DH 2 O baños. </li><li> Seco PGA andamios bajo el capó de cultivo de tejidos se sequen al aire durante 15 minutos con el ventilador. </li></ol> 3. Coser los brazos Dacron <ol><li> El uso de sutura Prolene 4.0 a coser pequeños trozos de manguitos de dacrón (1 cm) en cada extremo de la malla de la PGA con una superposición de 2 a 3 mm. Nota: no tener cuidado a la tubería de perforación de silicona (Figura 3). </li><li> Use la misma sutura para coser tres puntos de sutura alrededor del extremo libre de los manguitos de dacrón. Asegúrese de dejar suficiente suturas en los extremos libres de la sutura de Prolene de paso 5.1 (Figura 3). </li></ol> 4. Asamblea de biorreactor (día antes del comienzo de la cultura biorreactor) <ol><li> Remoje malla cosida y tubo de silicona, instrumentos quirúrgicos, y un alambre delgado en un baño de etanol al 70% durante 20-30 minutos. </li><li> Abrir las bolsas de autoclave que contiene el biorreactor y sumergirlo en un baño de etanol al 70% por lo menos 30 minutos antes del montaje. Asegúrese de lavar el biorreactor a fondo con etanol. La exposición de todos los componentes bio-reactor a 70% de etanol es una etapa de esterilización de adición. Este paso también elimina la endotoxina, que no se quita en autoclave y es perjudicial para las células vasculares. </li><li> Tire el tubo de silicona a través de armas de mano con alambre delgado para salir adelante. </li><li> Tome biorreactor de baño de etanol (mantener la malla sumergida en etanol). Fijar el andamio PGA dentro del biorreactor de Fijación de los puños Dacron sobre los labios de vidrio estalló apretando y atar las suturas de Prolene (Figura 3). </li><li> Conecte un extremo de los brazos laterales biorreactor para conectores a través de un tubo de silicona. </li><li> Tirar al otro lado del tubo de silicona con la suficiente tensión e inserte los otros dos conectores en el tubo de silicona. Mantenerse en el tubo de silicona con fuerza al insertar los conectores! </li><li> Vuelva a insertar biorreactor en el baño de etanol y lavar con etanol, tirando suavemente los conectores de los brazos laterales. </li><li> Voltear biorreactor más y permitir que un remojo durante 10 minutos. </li><li> Biorreactor voltear del lado derecho y en remojo durante 10 minutos adicionales. </li><li> Drenaje de todo el etanol. </li><li> Establecer tres grandes placas de Petri (10 cm) en serie y biorreactor a cabo en el plato central para mantener el exceso de etanol goteaba de los extremos de los tubos. </li><li> Ras biorreactor y la malla de la PGA con el agua de cultivo de tejidos utilizando una pipeta de 5 ml o 10 ml. También el tejido ras cultura del agua en el tubo de silicona. </li><li> Escurrir el exceso de agua en placas de Petri a ambos lados del biorreactor. </li><li> Biorreactor secar durante la noche en la capilla con ventilador encendido y apagado UV. </li><li> Notas adicionales: asegúrese de barra de agitación estéril en el biorreactor. Asegúrese de no &quot;libera&quot; a la bio-reactor a partir de ahora, para evitar la contaminación. Cortar un montón de tiras de parafilm y se ponga en remojo en un baño de etanol al 70% pequeñas (de gran placa de Petri funciona bien). </li></ol> 5. Día 1: Configuración del biorreactor <ol><li> Coloque una placa de Petri estéril sobre la abertura de cada biorreactor para proteger el interior PGA andamio de contaminantes. </li><li> Montar sistema de flujo de biorreactor, como se indica en la Figura 4 y parafina todas las juntas de conexión. <ol><li> Limpie los conectores primero con toallitas con alcohol. </li><li> Conecte el puerto de inyección de tercer brazo, sin uso de biorreactores. </li><li> Remueva el tubo de morsa y atar extremo azul de la dilución salina establecen tan cerca de la unión Y-como sea posible. Tire de la abrazadera del tubo en su lugar para asegurar que no se traslade el líquido a esta parte del tubo </li><li> Retire la bolsa de IV, y conectar la tubería de morsa (rojo) en el momento final de la bolsa de IV. Asegúrese de limpiar el puerto de inserción con un algodón con alcohol en primer lugar. </li><li> Adjuntar morsa a un lado del sistema de flujo (a través del tubo de extremo blanco). </li><li> Inserte llave de 3 vías en el sistema de flujo. </li><li> Retire el transductor de presión y conectar con llave de tres vías. </li><li> Conecte el otro extremo del transductor de presión para la apertura de media en la bolsa. </li></ol></li><li> Conectarel biorreactor para el sistema de flujo a través de la Y-unión. Use una jeringa de 60 ml para añadir 350 ml de una fungizone% (mezcla de 5 ml fungizone con 495ml de PBS) a la bolsa de IV. </li><li> Apriete la bolsa IV para limpiar el sistema de flujo mediante el ajuste de las llaves de paso para permitir el flujo de PBS. Nota: revisar el interior de biorreactor para asegurar que no haya fugas. </li><li> Vuelva a suspender 8 x 10 6 SMC (alrededor de una confluencia T75) en 1.25ml de mediano y de semillas en cada scafffold PGA. Asegúrese de que la suspensión de células ha sido uniformemente goteó en el PGA de malla de dacrón de unión, así como en la parte inferior de la PGA de malla. </li><li> Limpie el borde de biorreactor con un algodón con alcohol a través de rotación del biorreactor lado evitar flotando. </li><li> Tapón de silicona tapa de montaje (Figura 2) <ol><li> Pelar bolsa de autoclave de nuevo con cuidado, asegurándose de no exponer a fondo de la tapa. </li><li> Conecte el puerto de inyección al tubo de alimentación en el conector macho. </li><li> Adjuntar PTFE 0,20 micras filtros a cada uno de los tres puertos de aire. </li><li> Tenga cuidado de no exponer a / tocar el fondo de la tapa durante este proceso. </li><li> Parafilm el puerto de inyección. </li></ol></li><li> Inserte la tapa del tapón de silicona en biorreactor de vidrio y asegúrese de que la sonda de alimentación en el interior del bioreactor no toque los andamios sin semillas PGA. Parafilm alrededor de la tapa. </li><li> Lugar biorreactor con el sistema de flujo dentro de la incubadora (de lado) y rotar el biorreactor cada 5 minutos durante 25-30 minutos. </li><li> Llenar la cámara de biorreactor con 400 ml de nuestros medios de cultivo 4.10 como se describe en la (Tabla 1). Este medio de cultivo es &quot;óptima&quot; para las arterias porcinas de ingeniería. </li></ol> 6. Día 6-7: Conexión de la bomba, y la primera alimentación <ol><li> Crecen los andamios sin semillas estática sin ningún tipo de bombeo pulsátil a través del tubo de silicona durante 6-7 días. No hay necesidad de un cambio medio o los suplementos de vitamina C durante este tiempo. </li><li> Asegúrese de que no haya fugas de PBS o retorcimiento del tubo de flujo del sistema antes de encender la bomba. </li><li> Encienda la bomba del sistema de flujo y asegúrese de ajustar la configuración de la bomba para que la presión se lee aproximadamente 270/-30mmHg. </li><li> Presiones de registro diario en toda la cultura y mantener la presión en 270/-30mmHg. Transductor de presión se puede conectar a un ordenador para leer y controlar la presión. </li></ol> Primera alimentación <ol start="5"><li> Ensamble inyección de puerto y filtro de PTFE en la alimentación de tapas para tanto el cambio medio y medio con fines de residuos deposal. </li><li> Coloque el tubo de alimentación con firmeza en la bomba e introduzca un extremo al puerto de alimentación del reactor biológico y el otro extremo de la tapa de la alimentación. Asegúrese de limpiar los puertos de inyección con toallitas de etanol. </li><li> Use una bomba de doble dirección Masterflex para bombear 200 ml de medio. A continuación, utilice una sonda de alimentación a la bomba de nuevo 200 ml de medio fresco de nuevo en el biorreactor. Comience siempre con una velocidad muy lenta, sobre todo cuando se bombea de nuevo a medio y del biorreactor. </li><li> Cambio de medio y 2x/semana suplemento de ácido ascórbico. Para añadir ácido ascórbico, el uso de una jeringa 30 ml para extraer 25 ml de medio y colocarlo a un lado de la campana de los tejidos. Disolver 25 mg de ácido ascórbico en 5 ml de PBS y filtrarla a través de un filtro de 0,22 micras. En primer lugar se inyecta el ácido ascórbico estéril en el tubo de alimentación y volver a agregar el medio 25ml sacado antes. La receta es medio en la Tabla 1. </li></ol> 7. Los resultados representativos: <img src="/files/ftp_upload/2646/2646fig1.jpg" alt="Figura 1"/> Figura 1. Los tubos y conectores para el montaje del sistema de flujo se muestra más arriba. <img src="/files/ftp_upload/2646/2646fig2.jpg" alt="Figura 2"/> Figura 2. El tapón de silicona conjunto de la tapa se muestra arriba. <img src="/files/ftp_upload/2646/2646fig3.jpg" alt="Figura 3"/> Figura 3. Esquema de montaje biorreactor se muestra más arriba. En el interior de los puños Dacron biorreactor se fijan en los brazos de cristal con los nudos de sutura azul. <img src="/files/ftp_upload/2646/2646fig4.jpg" alt="Figura 4"/> Figura 4. Sistema de flujo conectada a un tubo y biorreactor se muestra arriba. L/S18 tubería será bombeada por una bomba Masterflex y así conducir la corriente. El transductor de presión se mide la presión antes de entrar en el biorreactor en el curso superior. <img src="/files/ftp_upload/2646/2646fig5.jpg" alt="Figura 5"/> Figura 5. Imagen del buque cosecha de ingeniería. Barcos de ingeniería que parecen ser opaco y alcanzar un espesor de pared de aproximadamente 250 micras después de un cultivo de 8 semanas en condiciones pulsátil. <img src="/files/ftp_upload/2646/2646fig6.jpg" alt="Figura 6"/> Figura 6. Hematoxilina y eosina secciones transversales de los vasos de ingeniería. A y B son de 8 semanas no pulsátil y los vasos impulsos, respectivamente.C y D son cuatro semanas de los buques no pulsado y pulsado, respectivamente. L indica el lado luminal de los vasos. La barra de escalas es 100μm. <img src="/files/ftp_upload/2646/2646fig7.jpg" alt="Figura 7"/> Figura 7. Manchas de tricrómico de Masson para el colágeno (azul) para las secciones transversales de los vasos de ingeniería. A y B son de 8 semanas de buques no pulsado y pulsado, respectivamente. C y D son cuatro semanas de los buques no pulsado y pulsado, respectivamente. Tenga en cuenta que el buque pulsos de 4 semanas muestra más colágeno que su contraparte no pulsátil. Las flechas blancas señalan fragmentos restantes del PGA en los vasos. La barra de escalas es 100μm. <img src="/files/ftp_upload/2646/2646fig8.jpg" alt="Figura 8"/> Figura 8. Tinción inmunoquímica de los marcadores de SMC en las arterias de la especie bovina de ingeniería. Músculo liso α-actina, calponin-1, y el músculo liso de la cadena pesada de miosina (SMMHC) son intermedios principios y finales de los marcadores contráctil SMC, respectivamente. Al final de la cultura de 12 semanas, las células de la pared del vaso expresar α-SM actina y cantidades moderadas de calponin-1 y SMMHC. La barra de escalas es 20μm. <table border="1"><tbody><tr><td> Componente </td><td> Cantidad </td></tr><tr><td> DMEM (DME / baja modificados) </td><td> 500 ml </td></tr><tr><td> FBS (suero fetal bovino) inactivado por calor </td><td> 100 ml </td></tr><tr><td> HEPES 1,0 M </td><td> 5 ml </td></tr><tr><td> La vitamina C (disuelto en PBS o DMEM) </td><td> 25 mg </td></tr><tr><td> Prolina / glicina / alanina 25 mg/25 mg/10 mg (disuelto en 5 ml de PBS) </td><td> 5 ml </td></tr><tr><td> CuSO4 1,5 mg (disuelto in1 ml de PBS) </td><td> 1 ml </td></tr><tr><td> Penicilina G en 10.000 unidades / ml </td><td> 5 ml </td></tr><tr><td> PDGF-BB (derivado de las plaquetas del factor de crecimiento-BB) en 10ng/ml </td><td> 5μg </td></tr><tr><td> bFGF (factor de crecimiento de fibroblastos) en 10ng/ml </td><td> 5μg </td></tr></tbody></table> Tabla 1. Los componentes de &quot;4.10&quot; medio se muestran en la tabla anterior. Con la excepción de PDGF-BB y bFGF, todos los demás componentes han de ser filtrada a través de un filtro de 0.2μm antes de su uso.

<ol>
	<li>Risau, W., Flamme, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenesis.">Vasculogenesis.</a> Annu. Rev. Cell Dev. Biol. 11, 73-91 (1995).</li><li>Fankhauser, F., Bebie, H., Kwasniewska, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influcence+of+mechanical+Forices+and+Flow+Mechanisms+on+Vessel+Occlusion.">The Influcence of mechanical Forices and Flow Mechanisms on Vessel Occlusion.</a> Lasers in Surgery and Medicine. 6, 530-532 (1987).</li><li>Niklason, L. E., Gao, J., Abbott, W. M., Hirschi, K. K., Houser, S., Marini, R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+arteries+grown+in+vitro.">Functional arteries grown in vitro.</a> Science. 284, 489-493 (1999).</li><li>Prabhakar, V., Grinstaff, M. W., Alarcon, J., Knors, C., Solan, A. K., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+porcine+arteries:+Effects+of+scaffold+modification.">Engineering porcine arteries: Effects of scaffold modification.</a> Journal of Biomedical Materials Research Part A. 67A, 303-311 (2003).</li><li>Mitchell, S. L., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+growing+tissue-engineered+vascular+grafts.">Requirements for growing tissue-engineered vascular grafts.</a> Cardiovascular Pathology. 12, 59-64 (2003).</li><li>Dahl, S. L. M., Rhim, C., Song, Y. C., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+compositions+of+tissue+engineered+and+native+arteries.">Mechanical properties and compositions of tissue engineered and native arteries.</a> Annals of Biomedical Engineering. 35, 348-355 (2007).</li><li>Quint, C., Kondo, Y., Manson, R. J., Lawson, J. H., Dardik, A., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tissue-engineered+blood+vessel+as+an+arterial+conduit.">Decellularized tissue-engineered blood vessel as an arterial conduit.</a> Proc Natl Acad Sci U S A. 108, 9214-9219 (2011).</li><li>Niklason, L. E., Yeh, A. T., Calle, E. A., Bai, Y., Valent&#237;n, A., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+Tools+for+Engineering+Collagenous+Tissues+Integrating+Bioreactors,+Intravital+Imaging,+and+Biomechanical+Modeling.">Enabling Tools for Engineering Collagenous Tissues Integrating Bioreactors, Intravital Imaging, and Biomechanical Modeling.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 3335-3339 (2010).</li><li>Gong, Z., Calkins, G., Cheng, E. -. c., Krause, D., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Culture+Medium+on+Smooth+Muscle+Cell+Differentiation+from+Human+Bone+Marrow-Derived+Mesenchymal+Stem+Cells.">Influence of Culture Medium on Smooth Muscle Cell Differentiation from Human Bone Marrow-Derived Mesenchymal Stem Cells.</a> Tissue Engineering Part A. 15, 319-330 (2009).</li><li>Gong, Z. D., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-diameter+human+vessel+wall+engineered+from+bone+marrow-derived+mesenchymal+stem+cells+(hMSCs.">Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs.</a> Faseb Journal. 22, 1635-1648 (2008).</li><li>Poh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+engineered+from+human+cells.">Blood vessels engineered from human cells.</a> Lancet. 365, 2122-2124 (2005).</li><li>American Heart Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biostatistical fact sheet: cardiovascular procedures. , (2002).</li></ol>

La calidad de los buques de ingeniería es en gran parte dictadas por la calidad de la SMC utilizados en el cultivo de tejidos. Los aspectos críticos del fenotipo contráctil SMC incluyen la morfología, el número de paso bajo, y la capacidad de proliferar en el interior del biorreactor. Se recomienda que el número de pases no será superior a P3 en el momento de la siembra de células en el andamiaje de polímero. Por otra parte, es crucial para confirmar que las fuentes de SMC son libres de micoplasma antes de su u...

<table border="1"><tbody><tr><th> Nombre del reactivo / Material </th><th> Proveedor </th><th> Número de catálogo </th></tr><tr><td> FBS (suero fetal bovino) inactivado por calor </td><td> HyClone </td><td> SH30071 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11885 </td></tr><tr><td> rhFGF-básica </td><td> R &amp; B </td><td> 234-FSE </td></tr><tr><td> rrPDGF-BB </td><td> R &amp; B </td><td> 520-BB </td></tr><tr><td> Penicilina G </td><td> Sigma </td><td> PENNA </td></tr><tr><td> De cobre (II) sulfato </td><td> Sigma </td><td> C8027 </td></tr><tr><td> Gylcine </td><td> Sigma </td><td> C8790 </td></tr><tr><td> L-alanina </td><td> Sigma </td><td> A7469-25G </td></tr><tr><td> L-prolina </td><td> Sigma </td><td> P5607-25G </td></tr><tr><td> Ácido Ascórbico </td><td> Sigma </td><td> A4544-25G </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375-100G </td></tr><tr><td> Tapón de silicona </td><td> Cole-Parmer </td><td> 06298-24 </td></tr><tr><td> Masterflex tubos L / S </td><td> Cole-Parmer </td><td> 06508-16, 06508-18 </td></tr><tr><td> Masterflex bomba </td><td> Cole-Parmer </td><td> 7553-80 </td></tr><tr><td> Dacron manguito </td><td> Maquet </td><td> 174406 </td></tr><tr><td> PGA se sintió </td><td> Concordia </td><td> MO000877-01 </td></tr><tr><td> 4-0 1.5 métricas Surgipro II sutura </td><td> Syneture </td><td> VP-557-X </td></tr><tr><td> 6-0 0.7 sutura Dexon métricas </td><td> Syneture </td><td> 7538-11 </td></tr><tr><td> 0.22μm PTFE filtros </td><td> Whatman </td><td> 6780-2502 </td></tr><tr><td> Three Way llave de paso </td><td> Edwards Lifesciences </td><td> 593WSC </td></tr><tr><td> Transductor de presión </td><td> Edwards Lifesciences </td><td> PX212 </td></tr><tr><td> IV bolsas </td><td> Baxter </td><td> R4R2110 </td></tr><tr><td> Conjunto de dilución salina </td><td> Flecha </td><td> W20030 </td></tr><tr><td> Tubo de silicona </td><td> Saint-Gobain </td><td> F05027 </td></tr></tbody></table>

engineering biological-based vascular grafts using a pulsatile bioreactor

Mucho esfuerzo se ha dedicado a desarrollar y fomentar la metodología de regeneración funcional de pequeño diámetro arterial pasa por alto. En el medio fisiológico, tanto la estimulación mecánica y química están obligados a mantener el adecuado desarrollo y la funcionalidad de 1,2 vasos arteriales. Sistemas de biorreactor de la cultura desarrollada por nuestro grupo están diseñados para apoyar la regeneración de vasos dentro de un control preciso quimio-mecánica entorno imitando la de los vasos nativos. Nuestra asamblea biorreactor y procedimientos de mantenimiento son bastante simples y altamente repetible 3,4. Las células musculares lisas (SMC) se siembran en un ácido poliglicólico tubular (PGA) de malla que se coloca en un tubo de silicona compatible y cultivadas en el biorreactor con o sin estimulación pulsátil de hasta 12 semanas. Hay cuatro características principales que distinguen a nuestro biorreactor de algunos predecesores. 1) A diferencia de otros sistemas de cultivo que simulan sólo la bioquímica de los vasos sanguíneos circundantes nativo, nuestro biorreactor también crea un ambiente pulsátil fisiológico mediante la aplicación de la tensión radial cíclico de los vasos en la cultura. 2) Varios barcos de ingeniería pueden ser cultivadas simultáneamente bajo diferentes condiciones mecánicas en un entorno químicas controladas. 3) El biorreactor permite una monocapa de células endoteliales (CE) para ser fácilmente cubierta en el lado luminal de los vasos diseñados para los modelos de implantación de los animales. 4) El biorreactor puede recipientes de cultivo diseñado con un tamaño diferente diámetro osciló entre 1 mm y 3 mm, ahorrando el esfuerzo de adaptar cada biorreactor individuales para ajustarse a un tamaño de diámetro específico. Los vasos de ingeniería cultivadas en nuestro biorreactor se asemejan a los vasos nativos sangre histológicamente en algún grado. Las células de la pared de los vasos maduros expresan marcadores de SMC contráctiles del músculo liso, como la cadena pesada de miosina (SMMHC) 3. Una cantidad sustancial de colágeno se deposita dentro de la matriz extracelular, que es responsable de la resistencia a la rotura mecánica de los vasos de ingeniería 5. El análisis bioquímico también indica que el contenido de colágeno de los vasos de ingeniería es comparable a la de las arterias nativas 6. Es importante destacar que el biorreactor pulsátil siempre ha regenerado los vasos que muestran propiedades mecánicas que permiten la implantación de experiencias exitosas en modelos animales 3,7. Además, este biorreactor se pueden modificar para permitir la evaluación en tiempo real y el seguimiento de la remodelación del colágeno a través del tiempo, de forma no invasiva, utilizando un microscopio óptico no lineal (NLOM) 8. Para concluir, este biorreactor debe servir como una plataforma excelente para el estudio de los mecanismos fundamentales que regulan la regeneración funcional de pequeño diámetro injertos vasculares.

Watch this Scientific Journal Video about Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor at JoVE.com

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Nuestro grupo ha desarrollado un sistema de cultivo bio-reactor que imita el estrés pulsátil fisiológicos del sistema cardiovascular para regenerar implantables de pequeño diámetro injertos vasculares.

La ingeniería biológica basada en los injertos vasculares El uso de un biorreactor pulsátil

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

engineering-biological-based-vascular-grafts-using-pulsatile

Research

JoVE Journal

Bioengineering

16.9K vistas.  Yale University. Nuestro grupo ha desarrollado un sistema de cultivo bio-reactor que imita el estrés pulsátil fisiológicos del sistema cardiovascular para regenerar implantables de pequeño diámetro injertos vasculares.

Video: Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Investigación

Bioingeniería

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

La operación de un biorreactor de sobremesa

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Construcción y caracterización de un Fold biorreactor Novela Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

El cultivo de células de mamífero utilizando un solo uso Sistema Neumático biorreactor

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Un biorreactor Novela de alta densidad de cultivo de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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.