The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

Los animales se mantuvieron en el Centro de Medicina Experimental (FEM, Charité, Berlín, Alemania), y todos los protocolos experimentales fueron revisados ​​y aprobados por la Oficina Estatal de Sanidad y Locales (LAGeSo, Berlín, Alemania; Reg. No. O 0365 / 11). Cosecha 1. Hígado <ol><li> Preparación prequirúrgica <ol><li> Utilice una placa de corcho para la fijación. Ponga un paño médica en el plato. Utilizando cuatro agujas, fijar una máscara de inhalación en la placa de corcho para la narcosis por inhalación intraoperatoria. </li></ol></li><li> Predisposición del Portal venosa cánula <ol><li> Conecte un catéter venoso periférico (G 20) a una llave de tres vías. El catéter venoso debe ser cortado oblicuamente, con una longitud de aprox. 1,5 cm. </li><li> De-aire del sistema usando una jeringa de 10 ml llena con solución Ringer. Es muy importante cuidadosamente-air de embolia de gas porque puede afectar negativamente a la descelularización perfusión. </li></ol></li><li> Predisposiciónde la cánula arterial <ol><li> Utilice una cánula de mariposa y cortar la aguja a una longitud de 5 mm. Colocar un tubo de 5 cm (ID 0,58 mm; OD 0.96 mm) a la aguja 5 mm y fijarlo con pegamento. Inserte un tubo de 2 cm de largo (ID 0,28 mm; OD 0.61 mm) en el tubo más grande y fijarlo con pegamento. </li><li> Slip otro tubo sobre esta construcción como tope y fijarlo con pegamento. De-aire de la cánula arterial con una jeringa de 5 ml llena con solución Ringer. </li></ol></li><li> Anestesia y Analgesia <ol><li> Inducir narcosis de la rata en la cámara de inducción de la anestesia por 3,5% de isoflurano en oxígeno a una velocidad de flujo de 1,5 L / min. </li><li> Para asegurar la analgesia segura durante la operación, inyecte metamizol (100 mg de masa corporal / kg) y dosis bajas de ketamina / medetomidina (10 mg / 0,1 mg por kg de masa corporal) por vía intraperitoneal. </li><li> Suministro de 1,5% de isoflurano en 1 L / min de oxígeno a través de la máscara de inhalación para su posterior mantenimiento de la anestesia quirúrgica. </li></ol></li><li> Arreglos preoperatorias &lt;ol&gt; <li> Afeitarse el abdomen liberalmente con una máquina de corte eléctrico. Humedecer el sitio de la incisión en el abdomen afeitado con etanol al 70% para la desinfección. </li><li> Colocar el animal en posición supina en el plato preparado, inserte la cabeza en la máscara de inhalación y fijar las extremidades con cinta y los pines médica. Retire el exceso de etanol y la piel con una compresa de gasa. </li></ol></li><li> Evaluación Anestesia <ol><li> Evaluar la profundidad de anestesia viendo la frecuencia respiratoria. Cuando la anestesia parece bastante profunda (40 a 60 respiraciones / min), comprobar si la analgesia es suficiente por tratar de desencadenar el reflejo pizca dedo utilizando pinzas quirúrgicas para pellizcar la piel entre los dedos de ambas extremidades posteriores. </li></ol></li><li> Enfoque abdominal <ol><li> Haga una incisión mediana en la pared abdominal caudal por encima de la vejiga. Cortar en este punto a los dos arcos costales laterales en forma en forma de V. Tenga cuidado de no cortar el diafragma. Tire de la pared abdominal cortar sobre el pecho. </li&gt; <li> Fijar el xifoides con una pinza Overholt, tire de él craneal y fijarlo con una banda elástica. Use hisopos de algodón mojado para evacuar el intestino. Envuelva el intestino en un vendaje de gasa húmeda. Por otra parte, cubrir todos los márgenes abdominales con vendajes húmedos. </li></ol></li><li> Preparación de hígado <ol><li> Diseccionar el ligamento falciforme. Use un vendaje de gasa húmeda para mantener los lóbulos hepáticos medial y craneal izquierda bajo la cúpula del diafragma. Exponer el ligamento hepatoduodenal y el lóbulo del hígado omental superior. </li><li> Utilice dos microforceps y tijeras de primavera para diseccionar cuidadosamente el ligamento unido al lóbulo omental superior hasta el lóbulo es móvil. Mueva el lóbulo usando hisopos de algodón mojado bajo la compresa de gasa, la celebración de los lóbulos medial y dejados en su lugar. </li><li> Después de la movilización del lóbulo hepático omental superior, mueva el estómago a la izquierda. Fijar el estómago con una pinza y diseccionar el epiplón menor con microforceps y las tijeras de primavera. </li><li> Movilizar el omentos menorl lóbulo hepático hasta que es móvil y posteriormente mover el lóbulo de nuevo en su cavidad. Utilice una abrazadera Backhaus para retener el duodeno. Mueva el duodeno a la izquierda para estirar y exponer el ligamento hepatoduodenal. Circunscribir el conducto biliar común en el ligamento hepatoduodenal estirada. </li><li> Diseccionar el conducto de la bilis desde el tejido adiposo y páncreas. Cortar el conducto biliar 1,5 cm de la bifurcación de la vía biliar. Diseccionar la vena portal desde el tejido adiposo circundante. Exponer la vena gastroduodenal. </li><li> Ligar la vena gastroduodenal dos veces con seda 6-0 y dividir la vena entre las 2 ligaduras. Diseccionar el tronco celíaco y la arteria hepática común del tejido circundante hasta que toda ramas arteriales se revelan. </li><li> Libera la arteria gastroduodenal del tejido circundante. Ate la arteria gastroduodenal dos veces con seda 6-0, con los lazos distantes unos de otros. Diseccionar la arteria gastroduodenal entre las 2 ligaduras. </li><li> Libera la arteria esplénicadel tejido circundante. Ate la arteria esplénica dos veces con seda 6-0, con los lazos distantes unos de otros. Diseccionar la arteria esplénica entre las 2 ligaduras. </li><li> Libera la arteria gástrica izquierda desde el tejido circundante. Ate la arteria gástrica izquierda dos veces con seda 6-0, con los lazos distantes unos de otros. Diseccionar la arteria entre las 2 ligaduras. </li><li> Circunscribir la vena cava inferior entre el riñón derecho y el lóbulo del hígado lateral derecha. Diseccionar la vena desde el espacio retroperitoneal. Exponer la aorta siguiendo el tronco celíaco. </li><li> Diseccionar el tejido adiposo retroperitoneal. Inyectar 1000 IE heparina en 1 ml de solución salina en la vena cava inferior. Retirar la cánula y cerrar la incisión con un algodón. Espere 1-2 min para el efecto sistémico de la heparina. </li><li> Circunscribir la aorta con pinzas. Diseccionar la aorta y Exsanguinate la rata. Además, diseccionar la vena cava inferior distalmente desde el riñón. </li></ol></li><li> Portal venosa Canulación (Use la cánula preparado en la sección 1.2) <ol><li> Haga una incisión peces aftosa en la vena porta distal. Abra la incisión con fórceps y canular la vena porta. Enjuague el hígado con 20 ml de solución de Ringer hasta que los decolora hígado y fijar la cánula con ligaduras (seda 6-0). </li></ol></li><li> Arterial canulación (Use la cánula preparado en la sección 1.3) <ol><li> Diseccionar la aorta por encima del tronco celíaco. Libera el segmento de aorta desde el espacio retroperitoneal. Cortar el segmento de aorta longitudinalmente para generar un parche aórtico. Inserte la cánula de construcción propia en un soporte estático. </li><li> Utilice 2 pinzas para deslizar el parche aórtico sobre la cánula. Ligar la arteria hepática con seda 6-0 sobre la cánula y fijar el parche en el pilar. </li></ol></li><li> Explantación Hígado <ol><li> Abra el diafragma, hacer una incisión circular y diseccionar el hígado a partir de las estructuras circundantes. </li></ol></li><li> Almacenamiento <ol><li> Store el hígado en un tanque de 500 ml estéril lleno de solución 350 ml de timbre hasta su uso. </li></ol></li></ol> 2. soluciones detergentes <ol><li> 10% de solución de Triton X-100 de stock <ol><li> Llene una botella de 1000 ml con 900 ml de agua destilada. y añadir 100 ml de Triton X-100 (99,9%). Utilice un agitador para disolver el Triton X-100. Llena la botella con agua destilada. hasta un volumen total de 1.000 ml se alcanza. </li></ol></li><li> 1% Triton X-100 <ol><li> Añadir 100 ml de la solución madre (10%) a una botella de 1000 ml. Añadir 900 ml de agua destilada. y agitar la botella dos veces. Filtrar la solución 1% de Triton X-100 a través de un filtro estéril. </li></ol></li><li> 10% de SDS de la Solución <ol><li> Llene una botella de 1000 ml con 900 ml de agua destilada. y añadir 66,99 g de SDS (99,9%, la densidad de 0,67). Utilice un agitador para disolver el SDS. Llena la botella con agua destilada. hasta un volumen total de 1.000 ml se alcanza. </li></ol></li><li> 1% de SDS <ol><li> Añadir 100 ml de la solución madre (10%) a un bot 1000 mltle. Añadir 900 ml de agua destilada. y agitar la botella dos veces. Se filtra la solución de SDS al 1% a través de un filtro estéril. </li></ol></li></ol> 3. Descelularización <ol><li> Puesta en marcha del dispositivo de perfusión <img alt="Figura 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> Figura 1:. Esquema del dispositivo de perfusión para Selectivo arterial y venoso portal perfusión de cuatro hígados de rata bajo oscilantes Condiciones Presión (. Modificado de Struecker et al 14) <ol><li> Enlace el drenaje a una llave de paso de tres vías y una extensión Heidelberger para regular el nivel de llenado y llenar el reactor con 500 ml de NaCl 0,9%. <img alt="Figura 2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> Figura 2:. Esquema de la perfusión set-up link la cámara de perfusión (1,400 cm 3) (1) al extremo distal (4) de la trampa de burbujas (5) a través del acceso venoso portal (2) o a través de la ac arterialproceso (3). La trampa de burbujas (5) debe estar equipado con una obturación (4) en el extremo distal y un orificio de ventilación (6). Conectar la trampa de burbujas (5) a una extensión de Heidelberger (7). Enlazar la extensión Heidelberger al segmento de bomba (8). Además, conectar el segmento de bomba (8) a otra extensión Heidelberger (9). Inserte la última extensión Heidelberger (9) en la botella de solución de detergente (10). Conectar el respirador (11) a la cámara de perfusión (o al distribuidor de presión en el caso de múltiples perfusiones). Para drenar los fluidos del reactor, conecte la salida de la botella de residuos (12). <ol><li> Introduce el segmento de la bomba en la bomba. Arrancar la bomba para llenar el ciclo con PBS, cerrar el extremo distal de la trampa de burbujas y abrir el respiradero. Cuando la parte inferior de la burbuja está cubierto de forma segura con PBS (2-3 cm), cerrar el respiradero y abrir el extremo distal de la trampa de burbujas. </li><li> Instalación de hígado <ol><li> Enlazar la llave de tres vías de la cánula de la vena portal para el acceso reactor relacionada. Connect la cánula arterial para el acceso arterial. Tenga cuidado para asegurar que el aire no entra en los sistemas. </li></ol></li><li> Aplicación de oscilantes Condiciones Presión <ol><li> Use un respirador con una frecuencia de 15 lpm y una amplitud de 26 mbar (min. 4 mbar, máx. 30 mbar). Conectar el respirador a la cámara de distribución de la presión y conectar la cámara al reactor. Iniciar la aplicación de presión. </li></ol></li><li> Protocolo Descelularización <ol><li> Realice los pasos de perfusión con un caudal de 5 ml / min. Iniciar la descelularización con la perfusión del hígado con 1% de Triton X-100 durante 90 min. Permitir emanación de residuos a través de la salida del dispositivo de perfusión para mantener el nivel de fluido dentro de la cámara de perfusión constante y por encima del hígado perfundido. Después de 90 min, cambiar el detergente a 1% de SDS. <img alt="Figura 3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="Mesa 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;p&gt; Figura 3: Protocolo de perfusión para todos los grupos experimentales.

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The authors have nothing to disclose.

Aunque la técnica presentada para la cosecha de hígado de rata y descelularización es fácilmente reproducible, hay ciertos pasos críticos a considerar: Durante la preparación para la cosecha del hígado, es importante para evitar el sangrado grave porque va a activar la coagulación de la sangre y puede conducir a la formación de coágulos de sangre dentro del hígado. En nuestra opinión, es ventajoso incisión en la aorta abdominal directamente antes de la canulación de la vena por...

Descelularización y recelularización pueden permitir la generación de órganos funcionales, trasplantables in vitro 1. Mediante la eliminación de las células y material antigénico (por ejemplo., ADN, los epítopos alfa-gal) de un órgano, la matriz extracelular no o menos inmunogénico (ECM) se puede obtener. Esta matriz conserva la microanatomía tridimensional de un órgano y puede servir como la biomatriz ideal para repoblación con células de un origen diferente, posiblemente xenogénico 2. Por lo tanto, una matriz de hígado de rata descelularizado podría ser repoblado con células de hígado humano. Este micro-hígado humanizado podría servir como un modelo ex vivo para la investigación de enfermedades (por ejemplo., Enfermedades metabólicas congénitas, enfermedades virales o tumores malignos) o para las pruebas farmacéuticas preclínico 3. Varios protocolos diferentes para perfusión descelularización hígado de rata ya se han publicado 4-13. En todos los protocolos, decellularzación se logró mediante la perfusión de detergentes iónicos o no iónicos alcalinas través de la vena portal canulado. A lo mejor de nuestro conocimiento, fuimos el primer grupo reportar rata descelularización hígado por perfusión selectiva a través de la vena porta y / o la rata arteria hepática 14. Activación de la perfusión selectiva de los diferentes sistemas vasculares en el hígado puede permitir mejores resultados descelularización y, además, pueden desempeñar un papel importante en la repoblación celular. En el estudio se detalla aquí, los hígados se perfundieron en un dispositivo de perfusión de propiedad a medida, permitiendo la perfusión bajo condiciones de presión oscilantes. Estas condiciones de presión imitan la perfusión respiratoria dependiente fisiológica del hígado: in situ, el hígado cuelga bajo la cópula del diafragma, cuyo movimiento durante la respiración tiene un impacto directo en la perfusión del hígado. La inspiración conduce específicamente a la reducción del diafragma y apretando del hígado, optimizandoflujo de salida hepato-venosa, mientras que la expiración conduce a la elevación del hígado y disminución de la presión intraabdominal para optimizar flujo de entrada venosa portal-15. Nuestro objetivo fue evaluar si oscilantes condiciones de presión tienen un impacto en la homogeneidad de la perfusión descelularización hígado de rata imitando condiciones intraabdominales ex vivo. La homogeneidad del proceso de descelularización puede ser un factor subestimado en descelularización perfusión. Todos los agentes conocidos utilizan para Descelularización hígado causan alteraciones en el ECM. Las células en áreas mal perfundidas permanecen dentro de la ECM, mientras que otras áreas están ya completamente descelularizado. Para disolver las células restantes, la duración de la perfusión o la presión deben ser elevados, causando más alteraciones a las áreas bien perfundidos. Por lo tanto, los detergentes para descelularización deben ser distribuidos de forma homogénea dentro del órgano.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
		</tr>
		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
		</tr>
		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
		</tr>
		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
		</tr>
		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
		</tr>
		<tr>
			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
		</tr>
		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
		</tr>
		<tr>
			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
		</tr>
		<tr>
			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
		</tr>
		<tr>
			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
		</tr>
		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
		</tr>
		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for decellularization of rat livers in an oscillating-pressure perfusion device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

La homogeneidad y por lo tanto la efectividad de diferentes protocolos de descelularización se evaluaron por observación macroscópica, el análisis histológico, y el análisis del contenido de ADN restante dentro de matrices de hígado descelularizados. Además, se realizó la colada de corrosión para visualizar la microanatomía de los hígados intactos después de descelularización. Macroscopía Durante descelularización, hígados convierten lucent, lo q...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedimiento para Descelularización de rata hígados en un dispositivo de perfusión oscilante presión

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K vistas.  Charité – Universitätsmedizin Berlin. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Video: Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Descelularización y recelularización de hígados enteros

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Investigación

Bioingeniería

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedimiento para descelularización de corazón porcino por perfusión coronaria retrógrada

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Instrumento ultrasónico para la conducción del nervio bloquear en modelos de rata diabética

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desarrollo de un Biosensor de ADN electroquímico para la detección de un patógeno de transmisión alimentaria

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dos métodos de descelularización de tejidos vegetales para aplicaciones de ingeniería de tejidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Una novedosa técnica quirúrgica como base para In Vivo parcial ingeniería hígado de rata

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Descelularización de todo humano corazón dentro de una bolsa presurizada en una orientación invertida

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Obtención y perfusión-descelularización de colgajos vascularizados porcinos en un biorreactor de perfusión personalizado

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Obtención y descelularización de extremidades posteriores de ratas utilizando un biorreactor basado en perfusión ex vivo para alotrasplante compuesto vascularizado

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...