Parte I. Preparación para la cirugía <ol><li> El día antes de la cirugía de la rata, asegúrese de que todas las soluciones se hacen, pero no le agregue nada enzimas todavía. Buffers A, B, y el tampón de lavado a todos debe ser filtrada para la esterilidad antes de su almacenamiento. Los buffers de todos deben ser almacenados a 4 ° C. </li><li> 6 y placas de cultivo de tejidos debe ser recubierto con 10-20 mg / ml de laminina en 2 ml 1XSFM el día anterior también, y deben ser almacenados a 37 º C en una incubadora de CO 2. </li><li> En el día de la cirugía, preparar los instrumentos quirúrgicos de esterilización de la pinza, tijeras, pinzas y con el 70% de etanol y dejar secar sobre una toalla de papel. </li><li> Una jeringa de 1 mL llena con 0,5 ml de solución de heparina (90 U / ml) se prepara. </li><li> Ahora es una buena práctica para lavar el aparato de perfusión mediante la ejecución a través de etanol al 70%, utilizando la bomba peristáltica a la inversa. Una vez terminado el enjuague con etanol, enjuagar el aparato con agua doblemente destilada, y por último enjuague con tampón A. El flujo a través se recoge en un recipiente colocado en un baño de agua 37 ° C. </li><li> Para preparar el aparato de perfusión limpiar, rellenar el tubo de la jeringa derecha con 40 ml de tampón A y el tubo de la jeringa izquierda con 40 ml de tampón B. </li><li> El primer tubo de la perfusión a la punta del catéter, permitiendo un Buffer de flujo a través del aparato. Rellenar el búfer de una jeringa con el nivel de 40 ml. Asegúrese de que no haya burbujas de aire atrapado en el tubo después de este paso. </li><li> Ahora, cierra el buffer de una válvula de llave de paso de la jeringa y repetir el proceso con Tampón B de tal manera que el tubo de perfusión está preparado con buffer B. </li><li> Finalmente, el aparato de perfusión se queda con la llave de paso del buffer B válvula abierta, pero con la corriente dejó de usar el regulador en la línea IV. </li><li> Deseche el tampón de flujo, aunque en el vaso de recolección. </li><li> La última cosa que hacer antes de la cirugía consiste en añadir las enzimas necesarias para su solución de reserva, y después se filtra la solución de la enzima al igual que las otras soluciones se filtraron el día anterior. Una vez filtrada, esta solución se puede dejar a temperatura ambiente. </li></ol> Parte II. La disección de corazón de rata <ol><li> Protocolo estándar después de la eutanasia a la rata con isoflurano en una cámara de gas. </li><li> Esterilizar el área de la incisión en el tórax con un 70% de etanol. </li><li> Pecho abierto con unas tijeras y exponer el corazón. </li><li> Inyectar ventrículo izquierdo con solución 0,5 ml de heparina (90 U / mL). </li><li> Quitar el corazón de gran parte de la aorta lugar intacto, en el hielo frío buffer B. </li></ol> Parte III. Miocitos aislamiento <ol><li> Un corazón de rata típica debe producir una elevada fracción de la barra en forma de los miocitos (en comparación con las células muertas redondeadas) si el procedimiento que sigue es bien respetado. </li><li> Para empezar, pinza la aorta con el catéter. La punta del catéter no debe ser llevado demasiado lejos en el corazón para garantizar una buena perfusión del corazón a través de la arteria coronaria. </li><li> Una vez fijada, la corbata de la aorta con el catéter con una sutura. </li><li> A continuación, abra el regulador de la línea IV para permitir una velocidad de goteo rápido (~ 60 gotas / min) y permitir que B Buffer para vaciar el corazón. </li><li> Durante el tampón de lavado B, use las tijeras pequeñas y pinzas para quitar las aurículas y los tejidos grasos o de los pulmones se aferran al corazón. </li><li> Después de Buffer B ha terminado, cambiar el flujo de tampón A. </li><li> Mientras que el tampón A se le permite fluir durante 5 minutos, varias tareas pequeñas deben realizarse rápidamente. </li><li> En primer lugar, calentar la solución de la enzima en un baño de agua a 37 ° C. </li><li> Finalmente, cuando se agota un Buffer de la jeringa (pero aún presente en el tubo), carga de 50 ml de la solución de la enzima en la jeringa del aparato de perfusión </li><li> Ahora es necesario desechar todo el flujo a través del vaso de recogida en el baño de agua (con pipeta), hasta que la solución de la enzima perfunde el corazón. </li><li> Tan pronto como la solución de la enzima perfunde el corazón, activar la bomba peristáltica que está configurado para transferir la solución de la enzima de la copa de recolección para llenar la jeringa A. </li><li> Deje que la solución de la enzima al flujo a través del corazón durante 10 minutos. Cuando el corazón se digiere, comienza a verse hinchada. </li><li> Añadir 37,5 l de 0,1 M CaCl 2 a la solución de la enzima en una jeringa para administrar una concentración efectiva de 0,1 mM Ca 2 +. </li><li> Después de 10 minutos aumenta la concentración de calcio de 0,2 mm mediante la adición de un adicional de 50 l de 0,1 M CaCl 2 a la jeringa A y dejar que la perfusión continuar durante otros 10 minutos. </li><li> Cortar los ventrículos y la transferencia de un pequeño vaso estéril que contiene 20 ml de solución de la enzima. </li><li> En el vaso de aumentar la concentración de calcio de 0,4 mm mediante la adición de 40 l más de 0,1 M CaCl 2. </li><li> Suavemente pelos en el corazón en 10 o más piezas con un par de pequeñas tijeras estériles. </li><li> Incubar el vaso durante 5 minutos a 37 ° C con suave balanceo. </li><li> Añadir 40 ml de 0,1 M CaCl 2 aobtener una concentración efectiva de 0,6 mM Ca 2 + y con cuidado triturar los pedazos del corazón con una pipeta de plástico de 5.3 veces. </li><li> Después de incubar durante 5 minutos a 37 ° C, añadir 40 ml de 0,1 M CaCl 2 y triturar suavemente como antes. </li><li> Separados digerido sola miocitos de no digerido del tejido conectivo mediante la filtración a través de una malla estéril micras 500. </li><li> Permitir que las células se asientan en un tubo de 50 ml durante 10 minutos a temperatura ambiente. </li><li> Desechar el sobrenadante con una pipeta. </li><li> Resuspender suavemente las células en tampón de lavado # 1 y permitir que las células que conformarse con 10-20 minutos a temperatura ambiente. </li><li> Mientras que las células se están estableciendo, tome una pequeña alícuota, y utilizar este tiempo como una oportunidad para evaluar la calidad y viabilidad de las células en suspensión bajo un microscopio invertido. Una buena preparación que tienen una alta proporción (&gt; 80%) de la varilla, como las células con estrías claras. </li><li> Una vez que las células se han asentado, desechar el sobrenadante y resuspender las células suavemente en tampón de lavado # 2. Vamos a colocar las células a temperatura ambiente, que a su vez debe tomar 10-20 minutos, y descartar el sobrenadante. </li><li> Repita el proceso de lavado, una vez más con el tampón de lavado # 3, y las células estarán listos para la cultura. </li></ol> Parte IV. Cultivos celulares de miocitos <ol><li> Resuspender suavemente las células en los medios de comunicación el 5% de suero. Antes de transferir las células a las placas de cultivo de tejidos, lavar los platos con 1x SFM. Transferencia de las células a una densidad deseada y se incuba en una CO 2 incubadora de cultivo de tejidos durante 3-5 horas. </li><li> Interruptor de los medios de comunicación a 1x SFM. </li><li> Las células pueden ser cultivadas hasta por cuatro días y usados ​​cuando sea requerido para realizar experimentos. </li></ol> Parte V. Representante Resultados / Resultados No debe haber más de un 70% los miocitos del corazón viven bajo microscopio invertido cuando el protocolo se hace correctamente. <img src="/files/ftp_upload/1308/fig1.jpg" alt="la figura 1" /> Tabla 1: Solución para la receta de una L 10 veces más Ca 2 + sin buffer KH: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> masa </td><td> La concentración final </td></tr><tr><td> NaCl </td><td> 68,96 g </td><td> 1180 mm </td></tr><tr><td> KCl </td><td> 3,58 g </td><td> 48 mM </td></tr><tr><td> HEPES </td><td> 59,58 g </td><td> 250 mM </td></tr><tr><td> K 2 HPO 4 </td><td> 2,85 g </td><td> 12,5 mM </td></tr><tr><td> MgSO4 </td><td> 3,08 g </td><td> 12,5 mM </td></tr></tbody></table> Tabla 2: Solución receta para el Buffer L 1 A: Añadir 1,98 g de glucosa a 100 ml de tampón 10X KH y llevar a un volumen final de 1 L. ajustar la osmolaridad a la condición de la fisiología cerca (~ 300) con glucosa. Llevar a pH 7,4 con NaOH. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> Masa </td><td> La concentración final </td></tr><tr><td> 10X Buffer KH </td><td> 100 ml </td><td> 100 ml </td></tr><tr><td> Glucosa </td><td> 1,98 g </td><td> 11 mM </td></tr><tr><td> Llevar a 1 L </td><td></td><td></td></tr><tr><td> Llevar a pH 7,4 con NaOH </td><td></td><td></td></tr><tr><td colspan="3"> Ajustar la osmolaridad a la condición fisiológica cerca (~ 300) con glucosa. </td></tr></tbody></table> Tabla 3: Solución receta para Buffers enzima <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> masa </td><td> La concentración final </td></tr><tr><td> Colagenasa tipo II </td><td> 25 mg </td><td> 0,05% (w / v) </td></tr><tr><td> Proteasa XIV </td><td> 10 mg </td><td> 0,02% (w / v) </td></tr><tr><td> BDM </td><td> 0,025 g </td><td> 5 mM </td></tr><tr><td> Carnitina </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> Taurina </td><td> 0,031 g </td><td> 5 mM </td></tr><tr><td> Ácido glutámico </td><td> 0,020 g </td><td> 2 mM </td></tr><tr><td> 0,1 M CaCl 2 </td><td> 12.5 uL </td><td> 25 uM </td></tr><tr><td> Tampón A </td><td></td><td> Llene hasta 50 ml </td></tr></tbody></table> Tabla 4: Solución receta para 25X BDM / Taurina / BSA (B / T / B) <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> masa </td><td> La concentración final </td></tr><tr><td> BDM </td><td> 0,076 g </td><td> 125 mM </td></tr><tr><td> Taurina </td><td> 0,094 g </td><td> 125 mM </td></tr><tr><td> BSA </td><td></td><td> 0,1% (w / v) </td></tr><tr><td> Tampón A </td><td></td><td> Relleno a 6 mL </td></tr></tbody></table> Tabla 5: Solución receta para el Tampón B y tampones de lavado <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Buffer: </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> Añadir: </td><td></td><td></td><td></td><td></td></tr><tr><td> 0,5 M CaCl 2 </td><td> 0,4 ml </td><td> 0,1 ml </td><td> 0.125 ml </td><td> 0,15 ml </td></tr><tr><td> La concentración final de CaCl2 </td><td> 1 mM </td><td> 1 mM </td><td> 1,25 mM </td><td> 1,5 mM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 mL </td><td> 2 mL </td><td> 2 mL </td></tr><tr><td> Buffer un volumen final de </td><td> 200 ml </td><td> 50 ml </td><td> 50 ml </td><td> 50 ml </td></tr></tbody></table> Tabla 6: Solución Receta para los medios de comunicación cultivo <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X medio libre de suero (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> La concentración final </td></tr><tr><td> (Después de la dilución) </td></tr><tr><td> Carnitina </td><td> 0,099 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Taurina </td><td> 0,063 g </td><td> 10 mM (5 mM) </td></tr><tr><td> La creatina </td><td> 0,075 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Antibiótico / antimicótico </td><td> 0,5 ml </td><td> 1% (0,5%) (v / v) </td></tr><tr><td> Los medios de comunicación 199 </td><td> 45 ml </td><td> Llene hasta 50 ml </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> La concentración final </td></tr><tr><td> 2X SFM </td><td> 25 ml </td><td> 1X </td></tr><tr><td> Los medios de comunicación 199 </td><td></td><td> Llene hasta 50 ml </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X 5% de los medios de comunicación en suero </td><td></td><td></td></tr><tr><td></td><td></td><td> La concentración final </td></tr><tr><td> 2X SFM </td><td> 25 ml </td><td> 1X </td></tr><tr><td> FBS </td><td> 2,5 ml </td><td> 5% (v / v) </td></tr><tr><td> Los medios de comunicación 199 </td><td></td><td> Llene hasta 50 ml </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

Un paso fundamental es la velocidad con la que se cuelga el corazón aislado hasta en el sistema de perfusión. La duración del período de digestión enzimática puede ser un poco diferente para cada rata. El ajuste depende de lo suave que el corazón se vuelve después del período normal de la digestión. La recuperación lenta de Ca 2 + después de la digestión de la enzima es esencial para la obtención de Ca 2 +-tolerantes las células sanas. De conejillo de ind...

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		<div>
		<table border="1">
		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
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<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of adult rat heart myocytes

Cultivadas primaria para adultos células cardiacas de roedores son un importante sistema modelo para la investigación cardiovascular. Sin embargo, el establecimiento de los miocitos sólida, viable adultos cultivados puede ser un desafío técnico, la tasa de limitación de paso para muchos investigadores. Aquí se describe un protocolo para obtener un alto rendimiento de los miocitos adultos de corazón de rata que se mantienen viables en cultivo durante varios días. El corazón es aislado y perfundido con colagenasa y la proteasa en condiciones de poca Ca<sup&gt; 2 +</sup&gt; Las condiciones para recuperar los miocitos individuales. California<sup&gt; 2 +</sup&gt; Tolerante a las células se obtienen por el aumento gradual en el extracelular de Ca<sup&gt; 2 +</sup&gt; Concentración en tres etapas de lavado posterior. Las células se filtran, se resuspendieron en medio de cultivo, y se colocaron en los recibos de laminina recubierto. Miocitos cultivados obtenidos mediante este protocolo son viables para un máximo de cuatro días y son adecuados para la mayoría de los experimentos incluyendo electrofisiología, bioquímica, biología molecular y de imagen.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

En este trabajo se describe la forma típica de aislar y cultivar el corazón de rata adulta miocitos. Colagenasa y la proteasa se utilizan para digerir y aislar a los miocitos individuales. Miocitos cultivadas seguir este protocolo cumplir con los requisitos de la mayoría de los experimentos.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.1K vistas.  Columbia University. En este trabajo se describe la forma típica de aislar y cultivar el corazón de rata adulta miocitos. Colagenasa y la proteasa se utilizan para digerir y aislar a los miocitos individuales. Miocitos cultivadas seguir este protocolo cumplir con los requisitos de la mayoría de los experimentos.

Video: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Cultura organotípicos de retina de un conejo adulto

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Investigación

Biología

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantación de la ingeniería tisular en el corazón de rata

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

El aislamiento y la manipulación genética de los miocitos cardiacos adultos de microscopía confocal

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

El establecimiento de cultivos primarios de fibroblastos de adultos de los roedores

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Preparación de corazón de rata altamente acoplado mitocondrias

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Los métodos para el aislamiento, cultivo y caracterización funcional de los miocitos del nodo sinusal ratones adultos

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Aislamiento simultáneo de cardiomiocitos de alta calidad, células endoteliales y fibroblastos de un corazón de rata adulto

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Cultivo primario de células adrenocorticales rata y análisis de funciones Steroidogenic

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...