Este trabajo fue apoyado por becas de la SNF [31003A-138528], la Octav y la Fundación Marcella Botnar, la Fundación Novartis y la Fundación Emma Muschamp. Agradecemos a Martine Lambelet, y Jean-Christophe Stehle por su excelente asistencia técnica.

El Comité Ético de la Universidad de Lausana aprobó los experimentos, que están en conformidad con los principios establecidos en la Declaración de Helsinki de 1975, revisada en 1983 para el uso de tejidos humanos. 1. humano vena safena Harvest <ol><li> Obtener segmentos excedentes de las venas safena humanos no varicosas de los pacientes sometidos a cirugía de bypass extremidad inferior por isquemia. En la sala de operaciones, desinfección de la pierna entera con una solución de yodo y la caída del paciente para exponer la pierna desde la ingle hasta el pie. </li><li> Hacer una incisión media desde la ingle hasta la rodilla (dejando la porción interrumpida de la piel). </li><li> Cosecha de la vena safena interna con un pedículo de los alrededores (técnica de no tocar) el tejido. Ramas laterales seguros de las venas con 4-0 corbatas de seda. Inmediatamente almacenar un mínimo de 9 cm de largo excedente de segmento de la vena safena mayor, con un diámetro externo de 2,5-4 mm a 4 ° C en un medio RPMI-1640 Tmedio lutamax, complementado con un 12,5% de suero bovino fetal y llevarla al laboratorio. </li></ol> 2. EVPS Diseño <ol><li> Se monta el equipo general que se muestra en la Figura 1. Autoclave todos los equipos y mantener todos los componentes en condiciones estériles. Además, asegúrese de que el sistema es resistente al agua y no se escape de productos químicos en el medio. Utilice polimetacrilato de metilo (PMMA-GS) para la cubierta. Acero (X5 Cr Ni 18 10) y el plástico de polioximetileno (POM) como el soporte vena. </li><li> Diseño de la cámara de perfusión a la geometría deseada para permitir la colocación de la vena y su conexión. Asegúrese de que la profundidad (o radio si se utiliza la construcción cilíndrica) es al menos 2,5 cm por lo que permite la flexión mínima y la dilatación del recipiente junto con la cobertura constante por los medios de cultivo (Figura 1). El sellado es un tema importante y es la razón se utiliza rectangular construcción PMMA-GS. </li><li> Diseñar el apoyo vena para la geometr deseaday. Para evitar torceduras vena o más de distensión, permitir el ajuste de longitud empujando o tirando (tornillo no puede ser utilizado para ese propósito, como la vena se retorció junto con el tornillo). NOTA: Una barra de acero llena conectados por 2 piezas deslizantes en forma de L que soportan los cilindros vena 2 (5 mm de diámetro para adaptarse a la embarcación) y la vena (Figura 1B y Figura 2) se utiliza aquí. </li><li> Diseño de la columna de presión, de manera que la &quot;presión de reposo&quot; aplicada al sistema es: p = 0-10 hx = ρ xg, donde p = presión (N / m 2, Pa) h = altura de la columna de fluido (m) ρ = densidad del líquido (kg / m 3) y g = la constante gravitacional (9,81 m / s 2). Diseño de cuatro conductos de conexión, de arriba a abajo: para aplicar presión, para la salida (de la vena), la entrada (en la vena) y para permitir el cambio medio. </li><li> Preparar el medio. Sobre la base de estudios previos 5,11-14, elija RPMI-1640 suplementado con Glutamax, 12.5% De suero de ternera fetal, y la solución de antibiótico-antimicótico al 1% (10.000 U / ml de penicilina G, más 10 mg / ml de sulfato de estreptomicina, además de 25 mg / ml de anfotericina B, además de 0,5 g / ml: gentamicina). Tensión de cizallamiento (SS) está dada por SS = 4 μQ / π r * 3 Q es el caudal (ml / seg), r el radio (cm) del segmento de la vena, y μ es la viscosidad del medio de perfusión. <ol><li> SS modular mediante el ajuste de la viscosidad mediante la adición de 70 kDa de dextrano. Medir la viscosidad con un viscosímetro. Aquí, agregar 8% 70 kDa dextrano para establecer SS a 9-15 dyn / cm 2. </li></ol></li><li> Establecer la bomba de engranajes para inducir una señal cardioide pulsátil de 60 impulsos / min y amplitud constante generar un flujo unidireccional de 150 ± 15 ml / min, independientemente de la presión aplicada en el sistema y controlado por un ordenador. Asegúrese de que el software de conducción integra adquisición constante y el seguimiento de las presiones, la velocidad del flujo, frecuencia del pulso, y la señal. Si lo desea, utilice una segunda bomba (no synchronized) para producir un no laminar, flujo turbulento. </li></ol> 3. Asamblea EVPS (Figura 1) <ol><li> Antes de empezar, asegúrese de que todo el equipo es estéril. Realice todos los siguientes pasos en la asepsia en una campana de flujo laminar. </li><li> Coloque la vena en una placa de Petri llena de medio. Use una hoja de bisturí y dividir la vena en 3 segmentos iguales. </li><li> Lavar inmediatamente un segmento en PBS. Dividir el segmento en 3 partes, fijar uno en formol para morfometría. Congelar los otros dos para la transcripción cuantitativa (RT-PCR) y proteínas (Western blot) análisis. Considere estos segmentos como control, la vena no perfundido. </li><li> Utilice los 2 segmentos restantes para la perfusión. <ol><li> Muy suavemente inyectar medio en la vena y determinar la dirección del flujo normal; en presencia de válvulas se invierte la vena. </li><li> Sellado de las venas es de suma importancia para el éxito experimental. Compruebe si hay fugas a través de colaterales. Asegure todas las fugas con 6-0 suturas de seda. </li> &lt;/ Ol&gt; </li><li> Conectar el segmento de vena entre los dos cilindros metálicos, uno de los extremos a la vez (2.3, Figura 1). Asegure los cilindros con Ethibon 3-0 alrededor de las muescas (Figura 1A y B). <ol><li> Coloque el segmento venoso entero en la cámara de perfusión previamente llenado con medio. Repita el mismo procedimiento para el segundo segmento. NOTA: Si no se sella correctamente la vena para el cilindro será una fuente de la fuga, requiere reintervención, y aumentar significativamente el riesgo de infección y fracaso experimental. </li></ol></li><li> Para reforzar (malla) el segundo segmento, liberar los dos cilindros (con la vena adjunto) a partir de las piezas en forma de L (2.3 y Figura 1). <ol><li> Sea amable y no toque la vena con cualquier instrumento. Deslizar la malla primero en el cilindro a continuación, en la vena. A empujones empuje / tirón conseguirá la malla en la vena. </li><li> Una vez que la malla cubre toda la superficie de la vena a asegurar la jvena acketed a los cilindros con Ethibon 3-0. </li><li> Volver a montar el compuesto vena / cilindro para el apoyo en forma de L y transferirla a la cámara de perfusión, previamente llenado con el medio. </li></ol></li><li> Conectar cada cilindro metálico (en-y salida) a un Y-separador utilizando tubo de silicona tratado con peróxido con un diámetro interno de 3,2 mm. </li><li> Conectar el divisor de flujo de salida a un segundo divisor de Y-utilizando el mismo tipo de tubo. De este Y-separador, utilizar un tubo para medir la presión de perfusión a través de ambos vasos. Conectar el otro de nuevo a la columna para formar un sistema de bucle cerrado (Figura 2). </li><li> Dentro de la incubadora, utilizar un tubo largo (de un metro de longitud) para conectar la columna de la presión a la cabeza de la bomba. </li><li> Completar la puesta en marcha mediante la conexión de la cabeza de la bomba a la entrada Y-splitter con otro tubo de gran longitud (Figura 1). </li></ol> 4. Venas Perfusión <ol><li> Después de la asamblea EVPS ha sido completed, llenar la columna con medio (permanecer por debajo del conducto de salida de la vena para permitir la recarga). Añadir más a medio en la columna hasta que el sistema esté lleno. Mover todo el sistema en la incubadora mantenida a 37 ± 0,1 ° C con un pH mantuvo constante a 7,40 ± 0,01 (utilizando un algoritmo de CO 2 / pH basado en la ecuación de Henderson-Hasselbach). </li><li> Llevar el cabezal de la bomba de engranajes fuera de la incubadora y conéctelo a la unidad de bomba de engranajes. Atornille las varillas para fijar el conjunto. </li><li> Encienda la alimentación de la bomba, asegúrese de que esté activado en el software de manejo y permite 5 min para el medio a ser distribuido por igual en todos los compartimentos. </li><li> Para controlar la presión, utilizar un monitoreo vía arterial. Conectar la salida de presión EVPS (que corresponde al catéter arterial) al transductor de presión vinculada a la computadora. <ol><li> Asegúrese de que el tubo está completamente lleno de medio y no contiene burbujas. De-burbuja el sistema de cultivo a través de la &quot;li arterialtubo ne &quot;(Figura 2). Preste atención a la pantalla y buscar una señal cardioide pulsátil de 60 impulsos / min de amplitud constante. En este punto, la presión media es de entre 0-10 mm Hg. Si la presión es &lt;0 y la columna se vacía progresivamente busque una fuga (vena colateral insuficiente o sello entre la vena y el tubo). </li></ol></li><li> Ajuste la presión mínima a 6 mm Hg para una prueba venosa o en 90 mm Hg durante una prueba arterial. En estas condiciones, un inyector de aire se aplica la presión requerida para la columna y el sistema. </li><li> Cambiar el medio cada 2 días utilizando el tubo conectado a la columna de presión. Para evitar daños en el cambio de presión, abrir el tapón de la primera columna. </li></ol> 5. La finalización de la perfusión <ol><li> Después de 3 o 7 días de la perfusión: tomar las EVPS fuera de la incubadora y desmontar las venas. Desechar el 5 mm proximal y distal de la vena termina adjunta al equipo. Cortar una, rin 5 mm de grosor central degs del segmento restante y fijar en formol (morfometría). Congele los fragmentos restantes y reducir a polvo para otros análisis moleculares. </li></ol>

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Los autores no tienen nada que revelar.

Este estudio revela un vivo sistema de perfusión venosa ex (EVPS) para llevar a cabo extensos estudios hemodinámicos en las venas humanas. Este sistema permite que las venas safenas de perfusión bajo parámetros hemodinámicos definidos en la ausencia de factores de crecimiento y agravantes inflamatorias liberadas por las células circulantes in vivo. Por lo tanto, proporciona una mejor comprensión de las vías subyacentes implicados en el control de IH en venas humanos injertos 5,11,...

Las enfermedades cardiovasculares son la principal causa de morbilidad y mortalidad en los países occidentales 1. A pesar de los avances en los tratamientos endovasculares, cirugía de bypass sigue siendo el pilar de las terapias actuales, por lo tanto más de medio millón de injertos de vena se realizan anualmente en los Estados Unidos. Sin embargo, a pesar de décadas de investigación, el 30-60% de los injertos de vena de la extremidad inferior fracasan en los primeros años, debido a la hiperplasia de la íntima (IH) 2. Las fuerzas mecánicas, estrés particularmente baja cizalladura (SS) y la tensión alta de la pared, son clave en el inicio y desarrollo de esta respuesta hiperplásica 3,4. Para abordar esta cuestión, un sistema de perfusión in vivo venas ex (EVPS) se generó a estudiar, en condiciones estrictamente controladas hemodinámicas (presión y el estrés de cizalla), el comportamiento de las venas safena humanos. En este estudio, después de la inserción en la circulación arterial-como, de alta presión (media = 100 mm Hg) era suficiente para estimular la proliferación y la migración de células musculares lisas en la capa intimal (IH) 5. Estudios de mamíferos han sugerido el uso de refuerzo externo como un método eficiente para apoyar la &quot;vena arterializada&quot; y contrarrestar la hemodinámica aguda cambia la vena se enfrenta una vez implantado en un entorno arterial. La malla impedido el exceso de distensión, aumento de la tensión de cizallamiento, y redujo tensión de la pared y, en consecuencia IH 6-10. Sin embargo, los mecanismos subyacentes y su aplicabilidad a las venas humanas en la mejora de la permeabilidad de derivación no se han caracterizado completamente. Nuestros EVPS se utilizó para comparar, en condiciones que imitan las alteraciones de una vena caras Una vez insertado en un régimen arterial (estrés de alto cizallamiento y presión), el comportamiento de las venas safenas humanos en la ausencia y presencia de una malla tubular de poliéster macroporoso externo. Al prevenir la remodelación patológica y IH, la malla proporciona evidencia de su potencial eficacia clínica 11 </sup&gt;. Este estudio 1) presenta un modelo ex vivo de las venas safena humanos perfusión bajo presión controlada y tensión de corte 2) demuestra que la malla de poliéster-macro porosa externa reduce IH y proporciona información crucial para su potencial aplicación clínica.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:371px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:144px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 - Glutamax</td>
			<td>Life Technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a name="RANGE!A3">Penicilline/Streptomycine/Fungizone</a></td>
			<td>Bioconcept</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran from Leuconostoc spp. 500 gr.</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tampon PBS CHUV pH 7.1-7.3 1 lt.</td>
			<td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td>
			<td>100 0 324 00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5m</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MF0037ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-splitter&#160;</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>Y-connector</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Culture dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430165-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gearing pump - Reglo-Z</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>SM 895&#160;</td>
			<td>&#160;App-Nr 03736-00194</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump Head</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MI0008&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring Kit TRANSPAC IV</td>
			<td>icumedical</td>
			<td>011-0J736-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL Syringes</td>
			<td>B. Braun Medical SA</td>
			<td>4612041-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Etibon 3-0 FS-2</td>
			<td>Ethicon- Johnson&#38;Johnson</td>
			<td>EH7346H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mesh ProVena 6-8mm</td>
			<td>B. Braun Medical SA</td>
			<td>1105012-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl: Sodium Chlorure Solution perfusion 0.9% (100 ml)</td>
			<td>B. Braun Medical SA</td>
			<td>534534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex L/S Standard Drive</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>7521-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6024 E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowmeter module</td>
			<td>Transonic Systems Inc.</td>
			<td>TS410 and T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopcock with 3-ways</td>
			<td>BD Connexta Luerlock</td>
			<td>394600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex Filter</td>
			<td>Milian</td>
			<td>SE2M229I04</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for human saphenous veins ex vivo perfusion and external reinforcement

El apoyo principal de las terapias contemporáneas para la enfermedad arterial oclusiva extensa es puente venoso. Sin embargo, su durabilidad se ve amenazada por la hiperplasia de la íntima (IH) que a la larga conduce a la oclusión del vaso y el fracaso del injerto. Las fuerzas mecánicas, esfuerzo de corte particularmente baja y alta tensión de la pared, se cree que para iniciar y sostener estos cambios celulares y moleculares, pero su contribución exacta aún no se ha desenredado. Para evaluar selectivamente el papel de la presión y la tensión de corte sobre la biología de IH, un sistema de perfusión ex vivo (EVPS) fue creado para perfundir segmentos de venas safena humanos bajo el régimen arterial (tensión alta cizalladura y alta presión). Otras innovaciones técnicas permitieron la perfusión simultánea de dos segmentos de la misma línea, uno reforzado con una malla externa. Las venas se recogieron usando una técnica de no tocar y transferidas inmediatamente al laboratorio para su montaje en el EVPS. Un segmento del aislador del reciénvena ATED no fue perfundido (control, día 0). Los otros dos segmentos fueron perfundidos durante un máximo de 7 días, uno está completamente cubierta con una malla externa 4 mm (diámetro). La presión, velocidad de flujo, y el pulso fueron controlados y ajustados para imitar las condiciones hemodinámicas que prevalecen en la arteria femoral de forma continua. Al término de la perfusión, las venas se desmontaron y se usaron para análisis histológico y molecular. En condiciones ex vivo, la perfusión de alta presión (arterial, = 100 mm Hg media) es suficiente para generar IH y remodelación de las venas humanas. Estas alteraciones se reducen en la presencia de una malla de poliéster externa.

El EVPS proporciona una valiosa herramienta para evaluar de forma independiente las fuerzas hemodinámicas en la vena safena humana injertos remodelación y IH. La figura 1 muestra la cámara de perfusión y el apoyo vena. En las figuras 1A y B, el apoyo vena antes (Figura 1 A) y después (Figura 1B) de montaje, respectivamente, se representa. Está compuesto (desde la parte superior a la parte inferior) de ...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Los mecanismos que conducen al desarrollo de hiperplasia de la íntima (IH) y la insuficiencia injerto de vena son aún poco conocidos. Este estudio describe un sistema ex vivo para perfundir venas humanos bajo flujo y presión controladas. Además, la eficiencia de refuerzo de malla externa para limitar el desarrollo de la IH se evaluó.

Procedimiento para safena humana Venas<em&gt; Ex Vivo</em&gt; Perfusión y refuerzo externo

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

procedure-for-human-saphenous-veins-ex-vivo-perfusion-external

Research

JoVE Journal

Medicine

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

11.1K vistas.  Brigham and Women's Hospital/Harvard Medical School.  Los mecanismos que conducen al desarrollo de hiperplasia de la íntima (IH) y la insuficiencia injerto de vena son aún poco conocidos. Este estudio describe un sistema ex vivo para perfundir venas humanos bajo flujo y presión controladas. Además, la eficiencia de refuerzo de malla externa para limitar el desarrollo de la IH se evaluó. 

Video: Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Técnica de Subnormothermic<em&gt; Ex Vivo</em&gt; Perfusión hepática para el Almacenamiento, Evaluación y Reparación de marginales injertos hepáticos

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Investigación

Medicina

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Método de aislados<em&gt; Ex Vivo</em&gt; Perfusión pulmonar en un modelo de rata: Lecciones Aprendidas de Desarrollo de un Programa EVLP Rata

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Humano Dupuytren<em&gt; Ex Vivo</em&gt; Cultura para el Estudio de miofibroblastos y matriz extracelular Interacciones

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

Ex Situ Normotérmica máquina de perfusión de hígados de donantes

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusión renal para la Preservación de riñón Injertos antes del trasplante

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generación de microtumores Uso Humano 3D Biogel sistema de cultivo y las células de glioblastoma derivados de los pacientes para Kinomic de perfiles y pruebas de respuesta a los fármacos

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Un pequeño modelo de la perfusión hepática normotérmica Ex Vivo

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Estudios de Optocardiografía y Electrofisiología de Corazones Perfundidos Ex Vivo Langendorff

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

Determinación y control de la salida de energía externa durante la propulsión regular de la silla de ruedas Handrim

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Procedimiento para safena humana Venas

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Normotérmica

Generación de microtumores Uso Humano 3D Biogel sistema de cultivo y las células de glioblastoma derivados de los pacientes para Kinomic de perfiles y pruebas de respuesta a los fármacos

Un pequeño modelo de la perfusión hepática normotérmica Ex Vivo

Estudios de Optocardiografía y Electrofisiología de Corazones Perfundidos Ex Vivo Langendorff

Determinación y control de la salida de energía externa durante la propulsión regular de la silla de ruedas Handrim