The authors would like to thank Hans-Joachim Schurek for invaluable scientific advice. The authors would like to thank Monique Carrel and Mich&#232;le Heidemeyer for excellent technical assistance, David Penton Ribas and Nourdine Faresse for a critical reading of the manuscript and Carsten Wagner and J&#252;rg Biber for the NaPi-2a antibody. This work was supported by the Swiss National Centre for Competence in Research "Kidney.CH" and by a project grant (310030_143929/1) from the Swiss National Science Foundation.

Todos los procedimientos con animales descritos en este manuscrito se realizaron de acuerdo con la legislación suiza y aprobados por la administración veterinaria del Cantón de Zurich, Suiza. 1. tampón de preparación de <ol><li> Preparar soluciones de 1 - 4 y la solución de la hormona antidiurética (ADH) (Tabla 1). </li><li> Preparar el tampón de diálisis (Tabla 1). NOTA: Este es el tampón utilizado como el tampón de diálisis durante la perfusión. Más tarde, los eritrocitos se diluyeron en este tampón para formar el líquido de perfusión final. </li><li> preparado de eritrocitos. <ol><li> Diluir 250 ml de concentrado de eritrocitos humanos (material ensayado obtenida del banco de sangre local) a 500 ml con tampón de diálisis. Se centrifuga a 2.000 g durante 8 min. Eliminar el tampón, teniendo cuidado de no eliminar los eritrocitos. Se repite 3 veces. </li></ol></li><li> Preparar el (albúmina de suero bovino; BSA) de albúmina de buffer. <ol><li> En 200 ml de dialysitampón s, se disuelven 44 g de BSA utilizando una barra de agitación. Se filtra la solución con papel de filtro. </li></ol></li><li> Preparar el líquido de perfusión. <ol><li> Filtrar los eritrocitos de la etapa 1.3.1 a través de papel de filtro en el tampón de BSA. Llenar hasta un volumen total de 800 ml con tampón de diálisis. NOTA: Este es el líquido de perfusión final. El hematocrito ahora debe estar entre 8 y 12%. El líquido de perfusión se puede almacenar durante un máximo de 12 horas a 4 ° C. </li></ol></li></ol> 2. Inicio de la diálisis y Oxigenación <ol><li> Encienda el baño de agua que rodea el depósito más grande de amortiguación, depósito de regulación más pequeña, y la cámara húmeda (una pequeña cámara, de doble pared llevó a 37 ° C y 100% de humedad para sostener más tarde el riñón) a 37 ° C (Figura 2) . </li><li> Llenar el depósito de regulación más grande con el tampón de diálisis y el depósito más pequeña con el perfundido. </li><li> Encienda el 5% de CO 2 O de entrada de gas 2/95% a tampón de diálisis. </li><li> Cambie en diálisis continua del líquido de perfusión frente al tampón de diálisis. Tenga cuidado de usar un tubo de diálisis de bajo flujo. Continúe con el Paso 3. </li></ol><img alt="Figura 2" src="/files/ftp_upload/54712/54712fig2.jpg" /> Figura 2: Dibujo esquemático del circuito de perfusión. Esquema muestra los componentes principales del circuito de perfusión y la dirección del flujo de tampón. Todos los componentes rodeadas de azul oscuro se mantienen a 37 ° C con un baño de agua / termostato. 1: Diálisis tampón de por lo menos 3 veces el volumen del tampón de perfusión se burbujea continuamente con 95% O2 / 5% de CO 2. 2: Diálisis tampón y tampón de perfusión se dializan de forma continua entre sí en un tubo de diálisis por una bomba de rodillos. 3: througho constante Debido a esta diálisis, el tampón de perfusión se enriquece con 9% de O2 / 5% de CO 2 y electrolitos niveles se mantienenperfusión ut. 4: una bomba de rodillos propulsa el tampón de perfusión hacia el riñón. 5: Un receptor de aire elimina ondas peristálticas y actúa como una trampa de burbujas. 6: El transductor de presión (conectado a 4. (bomba de rodillos) para mantener una presión continua al tiempo que permite el flujo libre alterna). 7: A lo largo de la perfusión, el riñón se mantiene en una cámara húmeda para la humedad del aire 100% y 37 ° C de temperatura de riñón. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> 3. Procedimiento quirúrgico Parte 1 (para un diagrama de todas las ligaduras, véase la Figura 3) Nota: Realizar todas las ligaduras utilizando 5-0 hilo quirúrgico. <ol><li> Anestesiar un ratón mediante inyección intraperitoneal (10 l / g de peso corporal, 20 mg / ml de ketamina y 1 mg / ml de xilazina disuelto en 0,9% NaCl). Confirmar suficiente profundidad de anesthEIAS ensayando la ausencia de reflejos del pie trasero. </li><li> Fijar el ratón en una posición supina en la cámara húmeda. Proteger los ojos con ungüento veterinario. Colocar una jeringa de 1 ml por debajo de la columna vertebral para elevar los vasos lumbares. </li><li> Realizar una laparotomía media de la cresta del pubis a la apertura del esternón primero la piel, los músculos abdominales, con unas tijeras. </li><li> Retire el intestino y lo coloca en el lado izquierdo del ratón lateral del abdomen. </li><li> Liberar la vejiga a partir de tejido conjuntivo y explorar ambos uréteres y la uretra. </li><li> Colocar una ligadura alrededor del uréter izquierdo (I ligadura). Cierralo. </li><li> Colocar una ligadura alrededor de la uretra (ligadura II). Cierralo. </li><li> Colocar una ligadura &quot;lazo&quot; alrededor de toda la vejiga (ligadura III). </li><li> Incisión en la vejiga 1 mm. </li><li> Canular la abertura con 2 cm PE 50 tubos. </li><li> Cerrar ligadura III alrededor de la tubería. </li><li> Cortar el uréter izquierdo y la uretra distal de las ligaduras. el bladder está ahora unido al uréter derecho solamente y se mueve libremente. </li><li> Desactive la aorta abdominal de tejido conectivo y grasa. </li><li> Colocar una ligadura mediados de aorta abdominal (IV ligadura). </li><li> Colocar una ligadura alrededor de la aorta por debajo del diafragma entre la arteria mesentérica superior y el tronco celíaco (V ligadura). </li><li> Colocar una ligadura alrededor de la arteria mesentérica superior (VI ligadura). </li><li> Colocar una ligadura aórtica directamente debajo de la derecha y por encima de la arteria renal izquierda (VII ligadura). </li><li> Colocar una ligadura alrededor de la vena caudal del paquete (cava) (VIII ligadura). Vaya al paso 4. </li></ol><img alt="figura 3" src="/files/ftp_upload/54712/54712fig3.jpg" /> Figura 3: Representación esquemática de las ligaduras colocadas durante la cirugía. Vista del abdomen abierto después de la laparotomía. El intestino se mueve hacia la izquierda. L y R indican el riñón izquierdo y derecho. lalíneas negras muestran el área de la respectiva ligadura. Las ligaduras se colocan primero y luego cerrados, según el orden indicado en el texto. X marca la ubicación de la incisión para la canulación de la aorta. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> 4. El cebado del circuito de perfusión <ol><li> Arrancar la bomba rotativa y llenar el tubo con líquido de perfusión. Tenga cuidado de vaciar todas las burbujas de aire. </li><li> Complete el dispositivo receptor de aire a aproximadamente a mitad de la altura de líquido de perfusión. </li><li> Calibrar el transductor de presión a 0 mm Hg cuando todos los tubos se llena y el flujo es 0. Mantenga la aguja de perfusión a nivel del riñón durante este tiempo. </li><li> Mantener el caudal a un nivel mínimo constante (0,6 ml / min) y vaya al paso 5. </li></ol> 5. Procedimiento quirúrgico Parte 2 <ol><li> Colocar una pinza de ligadura entre el IV y la ramificación de la izquierda renal arteria. </li><li> Hacer una pequeña incisión en la aorta caudal de ligadura IV, teniendo cuidado de no cortar la pared dorsal. </li><li> Dilatar la abertura en la aorta con un dilatador de vasos. </li><li> Canular la aorta con una aguja (2 cm de largo, tirado PE 50), empujando la punta justo a la abrazadera. </li><li> Abra la pinza. </li><li> Empuje la punta de la aguja cranealmente hasta que llega a la unión de la arteria renal derecha y la aorta. </li><li> Cerrar ligadura VII. </li><li> Cerrar ligadura IV. </li><li> Abre el cofre con las tijeras de disección por el diafragma. Con un solo corte, separar la aorta, vena cava, el corazón y los nervios vegetativos. Con este paso, el animal se sacrificó mediante exanguinación rápida bajo anestesia profunda continua. </li><li> Comience control de la presión de la bomba de perfusión. Mantener la presión media de entre 80 y 100 mmHg. </li><li> Cerrar V. ligadura </li><li> Cerrar ligadura VI. </li><li> Cerrar ligadura VIII. </li><li> Libre el riñón derecho de tissu conjuntivoE y su incorporación en la cápsula adiposa con unas tijeras. </li><li> Cortar la aorta proximal a la ligadura V. </li><li> Cortar la arteria mesentérica superior distal a la ligadura VI. </li><li> Cortar el paquete recipiente de riñón de apoyo a cabo, teniendo cuidado de no cortar en los propios vasos. </li><li> Cortar el hígado en la conexión con el riñón. Tener cuidado para liberar el riñón, pero dejar una pequeña parte de la adherente hígado a ella, de modo que la vena cava se mantiene abierta por el mismo. </li><li> Tome el paquete de riñón de ratón. Retire el ratón desde la cámara húmeda. </li><li> Colocar una ligadura &quot;lazo&quot; alrededor de la conexión del hígado y el riñón (IX ligadura). </li><li> Canular la vena cava con una línea venosa (2 cm PE 50). </li><li> Cerrar ligadura IX. La salida venosa a través de la línea venosa debe comenzar inmediatamente. </li><li> Cierre la cámara húmeda. </li></ol> 6. Los análisis intermedios <ol><li> Durante la siguiente hora, supervisar continuamente el flujo sanguíneo y intravasculla presión ar 15. Recoger el flujo venoso, que se puede utilizar para el análisis de, por ejemplo, la liberación de renina renal 7. Recoger la orina para el análisis de la concentración de electrolitos y el filtrado glomerular 14. Después de 1 hora de perfusión, los riñones pueden ser congeladas instantáneamente por el Western Blot o fijarse para obtener imágenes de los enfoques 16. </li></ol>

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The authors have no competing financial interests and nothing else to disclose.

El ratón aislados del riñón perfundido es una herramienta para el estudio de la función renal en un ambiente controlado ex vivo durante 1 hora, reduciendo la brecha entre los experimentos in vivo en animales intactos, lo que puede ser defectuoso por el impacto de numerosos factores sistémicos, y los experimentos in vitro en segmentos de la nefrona aisladas o células cultivadas, lo que necesariamente descuidar el impacto de la estructura de los órganos intactos en la función. H...

La perfusión aislada de órganos ha sido objeto de un esfuerzo continuo entre los fisiólogos durante muchas décadas 1. La técnica permite la función del órgano, sin influencias sistémicas como la hipertensión arterial, las hormonas, o los nervios, para ser estudiado. Carl Eduard Loebell se considera que es el primero que ha descrito la perfusión con éxito de un riñón aislado, en 1849 2. Desde entonces, el aparato de perfusión ha sufrido refinamiento significativo. Frey y Gruber introdujeron un pulmón artificial para las bombas de oxigenación y perfusión continua para pulsátiles 2. Mientras que los primeros investigadores estudiaron principalmente los riñones de los mamíferos grandes, a saber, cerdos y perros 2 3 -el primer informe del uso de riñones de rata, por Weiss et al. , Fue un hito en el estudio de la perfusión de los órganos mamífero pequeño 4. Schurek et al. informaron la necesidad de la adición de eritrocitos de mamíferos al perfundido si tubular renal suficienteoxigenación debía lograrse 5. Crítico para experimentos a largo plazo fue la introducción de la diálisis continua de la memoria intermedia por el mismo grupo de investigación 6. En 2003, Schweda et al. fueron los primeros en informar de un riñón aislado perfundido funcional del ratón (MIPK) 7, posteriormente refinado por Rahgozar et al. 18 y Lindell et al. 14. Aunque técnicamente más difícil que la rata aislado y perfundido de riñón, el uso de la MIPK lleva la ventaja de permitir el uso de una amplia gama de ratones genéticamente alterados. Este artículo presenta los detalles del método de los autores de la perfusión de los riñones de ratones aislados durante 1 hora. El método permite la evaluación continua de la tasa de flujo renal, la resistencia vascular, la liberación de hormonas, el análisis de gases en sangre, análisis de orina, y la aplicación de fármacos. Después del procedimiento, los riñones podrían ser procesados ​​para el análisis molecular y bioquímica, se fijarán para microscopía, otrasplantado en un ratón receptor (Figura 1). <img alt="Figura 1" src="/files/ftp_upload/54712/54712fig1.jpg" /> Figura 1: Visión general de la posible entrada / salida para el riñón aislado y perfundido. BGA: análisis de gases en sangre. <a href="http://ecsource.jove.com/files/ftp_upload/54712/54712fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> Esta técnica es probable que reciba una atención creciente en los próximos años, ya que muchas aplicaciones innovadoras se están discutiendo con el amanecer de la perfusión renal normothermic prolongado antes del trasplante (con o sin la aplicación de anti-rechazo o drogas genoma de edición) 8, 9, 10 , 11, la bioingeniería de los riñones enteros de andamios descelularizados 12, y la aplicación de altas dosis de tintes fluorescentes para la imagen multifotónica 13 </sup&gt;. También es un modelo ideal con el que estudiar el papel de los genes específicos durante la lesión renal aguda 14. Un protocolo paso a paso se da para permitir que otros laboratorios para realizar la perfusión aislada de riñón de ratón con éxito. En primer lugar, se especifica la composición y preparación de la memoria intermedia. A continuación, la cirugía se describe en detalle y se muestran los pasos críticos. En tercer lugar, se presentan los datos que son representativos de una preparación exitosa: el flujo sanguíneo renal, la resistencia vascular, la tasa de filtración glomerular, y el electrolito fraccionada mediciones funcionales de micrografías de viabilidad y electrónicas de transmisión de la morfología de los diferentes segmentos de la nefrona de los riñones perfundidos todos excreción- fijo después de 1 hora de perfusión.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Perfusion Circuit:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Moist chamber 834/8</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2901</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Cannular with basket and side port</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2947</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Thermostat TC120-ST5&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-4544</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">ISM 827/230V Roller Pump Reglo Analogue</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-0114</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 1L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3438</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Reservoir jacketed for buffer solution 0.5L</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3436</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Pressure Transducer APT300&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3862</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">TAM-D Plugsys Transducer</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-1793</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">SCP Plugsys servo controller</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-2806</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Windkessel&#160;</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3717</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">HSE-USB data acquisition</td>
			<td style="width:173px;">Harvard Apparatus/Hugo Sachs Elektronik GmbH</td>
			<td style="width:119px;">73-3330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Low-Flux Dialysator Diacap Polysulfone</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">7203525</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:447px;">PE-Tubing for aorta cannulation 1.19mm I.D. x 1.70mm O.D.</td>
			<td style="width:173px;">Scientific Commodities Inc.</td>
			<td style="width:119px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:447px;">Name</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Buffer reagents:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Aminoplasmal 10%</td>
			<td style="width:173px;">B.Braun</td>
			<td align="right" style="width:119px;">134518064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium pyruvate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2256-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-Glutamic acid monosodium salt hydrate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1626-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">L-(-)-Malic acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1125-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Sodium-L-Lactate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">L7022-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">alpha-Ketoglutaric acid sodium salt</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">K1875-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaHCO3</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">60130-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">U5378-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatinine</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4255-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td style="width:173px;">Roche</td>
			<td align="right" style="width:119px;">10835242001</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2 * 6H2O</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2393-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">D-Glucose</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2 * 6H2O&#160;</td>
			<td style="width:173px;">Riedel-de-Ha&#235;n</td>
			<td align="right" style="width:119px;">12074</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">NaH2PO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638-500G</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Na2HPO4</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0876-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Antidiuretic Hormone dDAVP</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;">V2013-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">FITC-Inulin</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Filter used for erythrocyte filtration</td>
			<td>Macherey-Nagel</td>
			<td style="width:119px;">MN 615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BGA Analysis:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ABL 80 flex</td>
			<td style="width:173px;">Radiometer Medical ApS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Electron Microscope:</td>
			<td style="width:173px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Philips CM100 TEM</td>
			<td style="width:173px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the mouse isolated perfused kidney technique

El aislado de riñón perfundido ratón (MIPK) es una técnica para mantener un riñón de ratón bajo condiciones ex vivo perfundidos y funcionales para 1 hr. Este es un requisito previo para el estudio de la fisiología del órgano aislado y para muchas aplicaciones innovadoras que pueden ser posibles en el futuro, incluyendo descelularización la perfusión de bioingeniería renal o la administración de anti-rechazo o drogas genoma de edición en altas dosis para cebar el riñón para el trasplante. Durante el tiempo de la perfusión, el riñón puede ser manipulado, la función renal puede ser evaluada, y diversos productos farmacéuticos administrado. Después del procedimiento, el riñón puede ser trasplantado o tratados con la biología molecular, análisis bioquímicos, o microscopía. Este artículo describe el líquido de perfusión y la técnica quirúrgica necesaria para la perfusión ex vivo de los riñones de ratones. Los detalles del aparato de perfusión y se dan los datos se presentan mostrando la vESPONSABILIDAD de la preparación del riñón: el flujo sanguíneo renal, la resistencia vascular, y de orina de datos como, micrografías electrónicas de transmisión funcionales de los diferentes segmentos de la nefrona como lecturas morfológicas, y transferencias Western de las proteínas de transporte de los diferentes segmentos de la nefrona como lectura molecular.

Con el método descrito, aislados riñones de ratón pueden permanecer viables durante al menos 1 hr. Hemos probado la viabilidad del tejido después de 1 hora de perfusión continua con el funcional (flujo sanguíneo renal y la resistencia vascular, el análisis de gases en sangre del flujo venoso, la tasa de filtración glomerular, urinaria fraccional de Na + y K + excreción, y la osmolaridad urinaria) y morfológica (electrónica de transmisión micro...

Watch this Scientific Journal Video about The Mouse Isolated Perfused Kidney Technique at JoVE.com

The Mouse Isolated Perfused Kidney Technique

El aislado de riñón perfundido ratón (MIPK) es una técnica para mantener un riñón de ratón bajo condiciones ex vivo perfundidos y funcionales para 1 hr. Los tampones y técnica quirúrgica se describen en detalle.

La Técnica del riñón perfundido aislado del ratón

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

the-mouse-isolated-perfused-kidney-technique

Research

JoVE Journal

Biology

21.5K vistas.  University of Zürich.  El aislado de riñón perfundido ratón (MIPK) es una técnica para mantener un riñón de ratón bajo condiciones ex vivo perfundidos y funcionales para 1 hr. Los tampones y técnica quirúrgica se describen en detalle. 

Video: The Mouse Isolated Perfused Kidney Technique

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Basada en el chip tridimensional de cultivo celular en perfundido Micro-biorreactores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Investigación

Biología

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Mapeo óptico de corazones de ratas Langendorff perfundidos

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterización de los aislados y ventilados, y pulmón de ratón instrumentado perfundidos con flujo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

El trasplante de células directamente en el riñón de adultos de pez cebra

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

La disección del riñón adultos de pez cebra

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parámetro de medición de la apertura del poro de transición de permeabilidad en el corazón del ratón mitocondrias aisladas

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

La cuantificación individual Microvessel Permeabilidad en aislado de rata Blood-perfundido Preparación de pulmón

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...