Los autores gracias Nadine Woitasky y Peter Volk para asistencia técnica. Además, los autores agradecen a la Sra. Claudia Lorenz (médico escritor, ACCEDIS) por su ayuda en la preparación del manuscrito. Este manuscrito fue apoyado financieramente por DFG (Schlu 324/7-1).

la investigación se lleva a cabo conforme a la guía para el cuidado y uso de animales de laboratorio publicados por nosotros Instituto Nacional de salud (NIH publicación no. 85-23, revisada en 1996). En general, ratas wistar macho de 3 a 4 meses de edad y con un peso promedio de 250-350 g se utilizan para este protocolo. Un corazón de rata es suficiente para 20 platos de cultura (1 mL por plato; diámetro interior: 35 mm) con una densidad celular aproximada de 1.5 x 10 4 células/1000 mm 2.
 1. preparación de medios y reactivos <ol><li>carnitina-creatina-taurina (CCT medio) Nota: CCT medio es un medio complejo basado en medio 199 con el agregado de creatina, carnitina y taurina.
	<ol><li>Preparar 1 L de medio 199: Añadir 3,6 g Hepes y mezcla durante 1 hora. Luego añadir 655,5 mg creatina (5 mM), 395,4 mg de carnitina (2 mM) y taurina mg 625,5 (5 mM). Carnitina y taurina cambian pH a &#60; 7. Con el fin de inhibir el crecimiento de células contaminantes, por ejemplo, las células endoteliales o los fibroblastos, añadir 10 μm citosina β-D-arabinofuranoside al medio. Ajustar el pH con NaOH (2 mM) filtro estéril y 7.4 el medio. Almacenar el medio CCT a 4 ° C.</li>
	</ol></li> <li>Medio de Powell <ol><li>para 1 medio L Powell, disolver 6,43 g NaCl (110 mM) con 0,19 g KCl (2,5 mM), 0.16 g KH 2 PO 4 (1,2 mM), 0,3 g de MgSO 4 7 H 2 O (1,2 mM), 5,96 g Hepes (25 mM) y 1,98 g D (+ )-Glucosa monohidrato (10 mM) en Aqua estéril. Ajustar el pH con NaOH (2 M) a 7,4 y estéril el filtro del medio. Medio de Powell tienda a 4 ° C.</li>
	</ol></li> <li>Cloruro de calcio (CaCl 2) <ol><li>preparar una solución de 100 mM CaCl 2 (50 mL) y preparar alícuotas conteniendo 500 μl de CaCl 2. Congelar en alícuotas a -20 ° C.</li>
	</ol></li> <li>Preparación de medio de cultivo <ol><li>Prepare tres medios de cultivo celular: pre-galjanoplastia medio, galjanoplastia de medio y medio de lavado. Utilizar el medio CCT como base para todos los tres medios (tabla 1). Calcular medio CCT con 1 mL por placa de cultivo. Por lo tanto, preparar medio CCT 20 mL para 20 platos de cultura (diámetro interior: 35 mm).</li>
		<li>De la célula cultura placas: cada placa de cultivo de células de la capa (diámetro interior: 35 mm) con 1 mL pre-galjanoplastia medio. Almacenar las placas recubiertas a 37 ° C durante la noche o durante al menos 2 h antes de usar.</li>
	</ol></li></ol> 2. Aislamiento de cardiomiocitos adultos <ol><li>sistema de preparación de Langendorff perfusión <ol><li>calor medio forro y lavado medio a 37 ° C. descongelar un tubo 500 μl de CaCl 2 y pesan de 25 mg de colagenasa.</li>
		<li>Ras del sistema de perfusión de Langendorff con aqua estéril, luego dejar medio Powell circulan el sistema por 5 min</li>
		<li>Llene el sistema de perfusión de Langendorff con 80 mL de medio de Powell sin cualquier aire burbujas y gas el medio con oxígeno del 95%.</li>
		<li>Preparar un tubo (50 mL) con 40 mL de medio de Powell, calentar a 37 ° C y gas con el oxígeno del 95%.</li>
		<li>Preparar un hilo de unos 25 cm de longitud para atar el corazón quitado a la cánula de.</li>
		<li>Desengrasar una cuchilla con alcohol (70% por volumen) y fijarlo a la picadora. Un disco de plástico de la abrazadera en la picadora.</li>
	</ol></li> <li>Extracción de corazón <ol><li>anestesiar una rata wistar macho con 4% a 5% isoflurano y sacrificio con la dislocación cervical. Abierto el abdomen detrás del arco costal con un corte abdominal y, con el mismo par de tijeras, corte a través del diafragma para abrir la cavidad torácica.</li>
		<li>Quitar el corazón, junto con el pulmón y timo, cortando por encima del timo muy craneal en la cavidad torácica. Transferir el material a una solución salina helada inmediatamente.</li>
		<li>Saque el pulmón y timo, el corazón con una tijera de disección (grande) y por fijación del material con pinzas para cápsula, transferir éste a una nueva solución salina.</li>
	</ol></li> <li>Aislamiento <ol><li>Eliminar exceso de tejido, como residuos de timo, la tráquea, la grasa y tejido conectivo del corazón usando fórceps cápsula y una tijera de disección (grande o pequeño). Descubrir la aorta y cortar con una tijera de disección (grande o pequeño) entre el primer y segundo lugar branchial arquitecto</li>
		<li>Iniciar el goteo del sistema de perfusión de Langendorff. Coloque el corazón en la cánula del sistema de perfusión de Langendorff y fijar primero con una pinza de cocodrilo y luego con el hilo preparado. Enjuague el corazón hasta que quede libre de sangre.</li>
		<li>Disolver 25 mg de colagenasa en el cálido medio de Powell 5-6 mL y añadir 12.5 μl CaCl 2 (30 μm).</li>
		<li>Cierre de circulación moviendo un embudo de cristal, que está conectado con el sistema de perfusión de Langendorff, sobre el corazón de goteo y añadir la colagenasa solucionado para el sistema de perfusión. Iniciar la perfusión de 25 min con una velocidad de caída de 1 gota por segundo. 
		Nota: Durante la perfusión, el corazón se hinche y conseguir un aspecto céreo.</li>
		<li>Detener la perfusión después de 25 minutos y retirar el corazón del sistema de perfusión de Langendorff. Quitar la aorta, aurícula y tejido conectivo del corazón y abrir los ventrículos izquierdos y derecho.</li>
		<li>Picar el corazón dos veces en un ángulo de 90° (ancho de corte: 0,7 mm, velocidad: 0.15 cm/s). Repita este proceso manualmente con dos bisturíes para 10 s cada lado.</li>
		<li>Transferir 12 mL del medio de perfusión a un tubo nuevo (50 mL). Vierta la mezcla de células en este medio y digerir las células por otros cinco minutos a 37 ° C. mezcla la solución cada minuto.</li>
		<li>Filtrar la solución con el corazón digerido a través de una malla de nylon (200 μm) en un tubo nuevo (50 mL).</li>
		<li>Centrifugar la solución filtrada a 29 x g durante 3 min descartar el sobrenadante y añada 6 mL caliente Powell medio incluyendo 12,5 μl CaCl 2 (250 μm) para el sedimento celulares. Resuspender el precipitado a través de movimientos de agitación suave. Centrifugar nuevamente a 29 x g durante 2 min descartar el sobrenadante y añadir 6 mL caliente medio Powell sustituido con 25 μl de CaCl 2 (500 μm). Disolver el precipitado de células a través de suaves movimientos de agitación y añadir 12 mL caliente Powell medio incluyendo 120 μl CaCl 2 (1 mM). Centrifugar por tercera vez en 16 x g durante 1 minuto. Otra vez, eliminar el sobrenadante.</li>
		<li>Mezclar el sedimento celular con el galjanoplastia precalentado medio.</li>
		<li>Retire placas previos medio placas de cultura. Transferir 1 mL de medio, incluyendo los cardiomiocitos aislados, a cada placa de cultivo de la galjanoplastia. Incubar los cardiomiocitos aislados fresca a 37 ° C por 1 h.</li>
		<li>Quitar el medio de placas de placas de. Añadir 1 mL de medio a cada placa de cultivo de lavado y almacenar las placas a 37 ° C hasta 6 días sin cambiar el medio.</li>
		<li>Para investigar la influencia de diferentes productos químicos y tratamientos en ARVC, primero actualizar chapado en medio medio de lavado y después añadiendo diferentes productos químicos. 
		Nota: La evaluación con microscopía de luz: con la preparación de cada célula, deben controlarse cardiomiocitos de 150 a 300 por día por microscopia ligera. Subdividir todo contado cardiomiocitos en grupos según su aspecto (por ejemplo, &#34; en forma &#34;, &#34; redondo abajo &#34;, &#34; separarse &#34;, y &#34; aspecto inusual &#34;). La categoría &#34; separarse &#34; incluye todos los cardiomiocitos con seudópodos como las estructuras. &#34; Aspecto inusual &#34; incluye todos ARVC con una superficie irregular y no detectable membrana intacta de la célula.</li>
	</ol></li></ol> 3. Ejemplo Experimentos <ol><li>de la fluorescencia de la inmunofluorescencia, tinción de cardiomiocitos adultos <ol><li>analizar morfológicas y estructurales las conversiones de ARVC durante el cultivo mediante microscopía láser confocal. Utilice Phalloidin-TRITC para investigar las estructuras de la F-actina en &#34; en forma &#34;, &#34; redondo abajo &#34;, y &#34; difundir &#34; ARVC. Realizar la coloración según el fabricante &#39; protocolo de s. Se da un ejemplo para la tinción de Phalloidin-TRITC en referencia Nippert et al. 11. con la tinción de fluorescencia de la inmunofluorescencia, las diferencias en el de - y re - differentiation del aparato contráctil en cardiomiocitos adultos cultivados entre tratamientos experimentales (por ejemplo, con Swiprosin-1, ionomycin) puede ser investigado.</li>
	</ol></li> <li>RT-PCR cuantitativa en tiempo real (qRT-PCR) <ol><li>realizar qRT-PCR para investigar cambios en la expresión del mRNA de genes diferentes (por ejemplo, OSM, Swiprosin-1, β-MHC) durante el cultivo de ARVC. Tamaño de muestra suficiente, utilizar platos más grandes de la cultura (diámetro interior: 60 mm) con 2 mL de volumen. ARVC de cinco platos de cultura produce una muestra. Realizar aislamiento del mRNA y la transformación del cDNA según el fabricante &#39; Protocolo s.</li>
	</ol></li> <li>Técnicas de inmunoblot <ol><li>realizar Western Blots para investigar cambios en la expresión de la proteína (por ejemplo, para Swiprosin-1) durante el cultivo de ARVC. Utilizar una placa de cultivo (1 mL) por ejemplo.</li>
	</ol>
	</li>
</ol>

<ol>
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Los resultados mostrados son parte de la tesis doctoral de Franziska Nippert.

El comportamiento de los cardiomiocitos adultos en vivo está influenciado por muchas interacciones con otras células (p. ej., neuronas, células endoteliales, fibroblastos, células inflamatorias) y el sincicio eléctrico que forman1. Por lo tanto, estudiando la adaptación de la tensión de cardiomiocitos adultos exclusivamente requiere el aislamiento y cultivo de ARVC. Los principales efectos de aislar y cultivar ARVC son: 1) desconexión de la matriz extracelular y célula-c...

Cardiomiocitos adultos en vivo funciona como un sincitio eléctrico basado en los contactos célula-célula entre miocitos. Además, están influenciadas por las adyacentes células como fibroblastos cardíacos, células endoteliales, neuronas y células inflamatorias1. Con el fin de estudiar la capacidad de cardiomiocitos para adaptar su organización intracelular alterado condiciones de carga, como se ve durante la hipertrofia cardiaca, que es un paso inicial hacia la insuficiencia cardíaca, el aislamiento y cultivo de rata adulta ventricular cardiomiocitos (ARVC) es necesario2,3,4. Históricamente, cardiomiocitos primero fueron aislados de chick embrionario corazones5,6. Unos años más tarde, el primer aislamiento de cardiomiocitos terminalmente diferenciadas fue descrito por medio del agotamiento de calcio7. Sin embargo, estos cardiomiocitos adultos no eran tolerantes de calcio y por lo tanto no se podían utilizar para ensayos funcionales. Finalmente, en 1976 un nuevo protocolo permitió a Powell y toque investigar cardiomiocitos ventriculares adultos bajo condiciones fisiológicas8. Como primer paso, aislaron cardiomiocitos adultos bajo concentraciones bajas de calcio y posteriormente aumento calcio a concentraciones fisiológicas en un procedimiento paso a paso. Hoy, mayoría de los protocolos para el aislamiento y cultivo de cardiomiocitos adultos trabaja con este protocolo de calcio y uso de colagenasa para la digestión enzimática de la célula densa contactos1.
Para un exitoso cultivo, suero fetal de ternero (FCS) u Oncostatina M (OSM) se requiere. ARVC realizar un de - y re - differentiation con extensos cambios estructurales incluyendo sarcómero desmontaje y reforma9,10,11,12. Este proceso es acompañado por una nueva expresión de los genes de tipo fetal, como la cadena pesada de miosina β (β-MHC), como la hipertrofia y formación de seudópodos-como las estructuras, también llamado extensión de4,11, 13. Además, swiprosin-1 (EFHD2), una proteína recientemente identificada, desempeña un papel importante en el proceso de rediferenciación de ARVC cultivadas11. Como resultado, ARVC en cultura se transforman en células generalizadas, polimorfas, que espontáneamente muestran contracciones después de dos a tres semanas de cultura2,4,14.
Recientes descubrimientos han revelado que cardiaca differentiation re y de como ocurre bajo condiciones de cultivo imita características vistos en vivo durante la remodelación cardiaca10,15. Remodelación cardiaca es un proceso clave en enfermedades cardíacas16. Enfermedades cardíacas siguen siendo la principal causa de muerte en las sociedades industrializadas, una mejor comprensión de la biología de cardiomiocitos adultos es importante (que; 2015). Aislamiento y cultivo de ARVC pueden ayudar a desarrollar nuevas estrategias y medicamentos para el tratamiento de enfermedades cardíacas. Con este manuscrito, se proporciona un protocolo para el aislamiento y cultivo de ARVC. Además, se destacan algunas partes esenciales de este método en la sección de discusión.

<table><tbody><tr><td>Langendorff perfusion system</td><td>inhouse construction</td><td></td><td>double-walled with a water&lt;br/&gt; based heating system</td></tr><tr><td>Tissue chopper Mc Ilwain</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Aortic Cannula, OD 1,8 mm</td><td>inhouse construction</td><td></td><td></td></tr><tr><td>Abdominal shears</td><td>Aeskulap</td><td>BC772R</td><td></td></tr><tr><td>Capsule forceps</td><td>Eickemeyer</td><td>171307</td><td></td></tr><tr><td>Dissecting scissor large</td><td>Aeskulap</td><td>BC562R</td><td></td></tr><tr><td>Dissecting scissor small</td><td>Aeskulap</td><td>BC163R</td><td></td></tr><tr><td>Mash with Polyamid</td><td>Neolab</td><td>4-1413</td><td>mash size 200 &amp;mu;m</td></tr><tr><td>plastic disc</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Collagenase Typ II</td><td>Worthington</td><td>LS004177</td><td></td></tr></tbody></table>

isolation and cultivation of adult rat cardiomyocytes

En un corazón intacto, las células adyacentes influyen en cardiomiocitos adultos. Con el método de aislamiento y cultivo de cardiomiocitos adultos, es posible una investigación precisa del comportamiento de estas células en tratamientos específicos y ambientes. Este manuscrito presenta un protocolo para acertado aislamiento y cultivo de cardiomiocitos ventriculares de rata adulta (ARVC).
La rata se sacrificaron mediante dislocación cervical bajo anestesia profunda. A continuación, se extrae el corazón y la aorta se destapa. Posteriormente, se realiza la perfusión en el sistema de perfusión de Langendorff con el agotamiento de calcio y tratamiento de la colagenasa. Luego, tejido ventricular obtiene picada, recircula y filtrado, seguido de tres pasos de centrifugación con incorporación gradual de CaCl2 hasta que se alcanza la concentración fisiológica de calcio. ARVC se platean en platos de cultivo celular. Después de actualizar el medio de cultivo celular, ARVC pueden cultivar hasta seis días sin cambiar el medio de cultivo que contiene el suero. Aislamiento de ARVC es un proceso sensible del calcio. Pequeños cambios en la concentración de calcio intracelular provocar una disminución en la calidad y viabilidad de las células aisladas.
Recién aislado ARVC son en forma de barra. En los primeros días de cultivo pierden las forma morfología y forma seudópodos-como las estructuras (separarse). Durante esta formación morfológica ARVC degradan inicialmente sus elementos contráctiles seguidas por una reforma a través de fibras de actina estrés y sarcomerogenesis de novo . Después de una semana de cultivo, la mayoría ARVC muestran un aspecto generalizado con una estriación transversal claramente perceptible. Este proceso es sensible a la concentración de calcio intracelular, como tratamiento con ionomycin atenúa separarse. Los marcadores claves en este proceso de - y re - differentiation son cadena pesada de miosina β (β-MHC), Oncostatina M (OSM) y swiprosin-1 (EFHD2). Estudios recientes han sugerido que cardiaco re y de differentiation que ocurre bajo condiciones de cultivo imita características vistos en vivo durante la remodelación cardiaca. Por lo tanto, el aislamiento y cultivo de ARVC juegan un papel clave en la comprensión de la biología de cardiomiocitos.

Cardiomiocitos adultos en la cultura: La figura 1 muestra un resumen de cardiomiocitos adultos recién aislado 2 h después del último lavado. Aproximadamente el 75% de los cardiomiocitos tenía una morfología en forma de barra. El 25% restante demostrado un aspecto inusual con una morfología circular y no detectable intacto membrana de la célula (figura 1). Al final del cultivo (día 6), hasta 15% de los car...

Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

Isolation and Cultivation of Adult Rat Cardiomyocytes

Aquí, presentamos un protocolo para el aislamiento y cultivo de cardiomiocitos ventriculares de rata adulta (ARVC). ARVC aislada puede utilizarse para el cultivo de corto y largo plazo. El aislamiento y cultivo de ARVC pueden desempeñar un papel clave en el desarrollo de nuevos regímenes de tratamiento para enfermedades cardíacas.

Aislamiento y cultivo de cardiomiocitos de rata adulta

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

isolation-and-cultivation-of-adult-rat-cardiomyocytes

Research

JoVE Journal

Medicine

15.5K vistas.  Justus Liebig University Giessen. Aquí, presentamos un protocolo para el aislamiento y cultivo de cardiomiocitos ventriculares de rata adulta (ARVC). ARVC aislada puede utilizarse para el cultivo de corto y largo plazo. El aislamiento y cultivo de ARVC pueden desempeñar un papel clave en el desarrollo de nuevos regímenes de tratamiento para enfermedades cardíacas.

Video: Isolation and Cultivation of Adult Rat Cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

El aislamiento y criopreservación de rata neonatal cardiomiocitos

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Investigación

Biología del Desarrollo

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

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 ...

Biología

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 ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Aislamiento, cultivo y caracterización funcional de ratón adulto Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

Aislamiento de cardiomiocitos auricular de un modelo de rata de la falta de corazón relacionados con el síndrome metabólico con fracción de eyección preservada

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Medicina

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Aislamiento y cultivo de ratón adulto cardiomiocitos de Señalización Celular y<em&gt; In vitro</em&gt; Hipertrofia Cardíaca

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Aislamiento, cultivo y la transducción de cardiomiocitos de ratones adultos

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

Un método simple y efectivo para aislar consistentemente cardiomiocitos de ratón

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

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 ...