Este trabajo fue apoyado por fondos de Duke-NUS Graduate Escuela de Medicina de Singapur, Goh fundación y comunión Singapur NRF (NRF-NRFF2016-01) para Manvendra K. Singh.

Todos los experimentos fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales de Duke-NUS Graduate Medical School. 1. Recuperación de la embrionaria Ventrculos <ol><li> Sacrificar un ratón embarazadas transitoria en la etapa embrionaria deseada (E11.5 E12.5 o) usando una cámara de eutanasia con el suministro de gas de dióxido de carbono u otro método de eutanasia aprobada. </li><li> Coloca el ratón sobre su espalda en una mesa de disección. Desinfectar el abdomen de la hembra con un 70% de etanol. Levante la piel sobre el vientre y hacer una pequeña incisión (1-2 cm) con una cuchilla, a continuación, mantenga la piel con ambas manos y tire de él para exponer la pared abdominal. </li><li> Ahora corte de la pared abdominal para exponer la trompa uterina. El uso de fórceps estériles para levantar el cuerno uterino y corte con cuidado los tejidos grasos y los vasos sanguíneos unidos a la trompa uterina. Cortar el cuello del útero para recuperar el útero. </li><li> Coloque el cuerno uterino en una placa de Petri que contiene fosfato estéril fría 1x Buff<html> Ered salino (PBS) y enjuagar suavemente. Coloque la placa de Petri en el hielo. </li><li> Use las tijeras para cortar a través de la línea media del cuello uterino (opuesta al sitio donde se encuentra la placenta) para exponer los embriones que aún se encuentran en el interior del saco vitelino y pegado a la pared uterina a través de la placenta. </li><li> El uso de pinzas estériles, cortar abierto el saco vitelino embrionario para liberar al embrión. </li><li> Coloque el embrión en una nueva placa de Petri que contiene estéril frío 1x PBS. Decapitar al embrión y colocarlo en su parte posterior. A cielo abierto de la pared torácica para exponer el corazón. </li><li> El uso de fórceps estériles para levantar el corazón y cortar los vasos alrededor de ella para liberar el corazón de la pared torácica. Prestar especial atención para no dañar el epicardio del corazón con las herramientas quirúrgicas. </li><li> Se quita el tracto de salida y ambas aurículas. Transferir el ventrículo a otro plato con 1x PBS frío. Coloque el plato en el hielo. </li><li> Repita los pasos 1.6-1.9 hasta que todos los ventrículos se recuperan. </li></ol>Le &quot;&gt; 2. epicárdica explante Cultura Nota: Cultura estos explantes epicárdicas ya sea en la cámara de diapositivas de vidrio o en gel de colágeno para aplicaciones posteriores. <ol><li> Cámara de vidrio diapositivas <ol><li> Para preparar el medio de cultivo, añadir 10% de suero bovino fetal (FBS), 1% de penicilina / estreptomicina y 2 ng / ml de factor de crecimiento de fibroblastos recombinante 2 (FGF2) para el medio de Eagle modificado por Dulbecco (DMEM). Realizar todos los pasos de la campana de flujo laminar. </li><li> Añadir 500 l de medios de cultivo a cada pocillo de una cámara de 8 pocillos. </li><li> Coloque cada ventrículo escindido en el centro de un pozo. Oriente el ventrículo para mantener el lado diseccionado hacia abajo en la superficie inferior del pozo. </li><li> Introduzca suavemente la placa en un 37 ° C, 5% de CO 2 incubadora de cultivo celular. </li><li> Mantener el cultivo con el mínimo de perturbación para permitir que los explantes se adhieran. La formación de la monocapa epicárdica puede ser observado después de 48 a 72 hr. </li><li> Para la diferenciación de epicardial células en células musculares lisas, cultivar las células epicárdicas monocapa en medio de diferenciación para otros 6 días. Para preparar el medio de diferenciación, añadir 10% de FBS, 1% de penicilina / estreptomicina y 50 ng / ml de factor de crecimiento transformante beta recombinante 1 (TGF-β1) a DMEM. </li><li> Cambiar el medio de cultivo en días alternos. </li></ol></li><li> Gel de colágeno <ol><li> Preparar el gel de colágeno en una placa de 96 pocillos usando un kit comercial Cultura 3D colágeno de acuerdo con el protocolo del fabricante. </li><li> Para preparar el volumen deseado de gel de colágeno, se mezcla la cantidad apropiada de solución de colágeno con 5x de DMEM y la pipeta hacia arriba y abajo. La solución debe tornarse amarilla. </li><li> Añadir un volumen correspondiente de la solución de neutralización y mezclar bien inmediatamente pipeteando arriba y abajo. La solución se vuelve rosada. </li><li> Pipetear 100 l de esta mezcla en cada pocillo de una placa de 96 pocillos. Colocar la placa en un 37 ° C, 5% de CO2 durante 30-60 minutos a Albajo el gel se polimerice. </li><li> Añadir 200 l de medios de cultivo descritos anteriormente a cada pocillo. </li><li> Coloque cada ventrículo escindido en el centro de un pozo. Oriente el ventrículo para mantener el lado diseccionado hacia abajo en el gel de colágeno (ápex del ventrículo debe enfrentar el experimentador). Introduzca suavemente la placa posterior en la incubadora de cultivo celular. </li><li> Después de 3 días, retire los ventrículos. Coloque la placa de nuevo en la incubadora de cultivo celular durante otros 2 días. </li><li> Cambiar el medio de cultivo en días alternos. </li></ol></li></ol> 3. Fijación y Visualización Nota: las células epicárdicas pueden visualizarse en cualquiera de portaobjetos de cámara de vidrio o gel de colágeno. <ol><li> Aspirar los medios de cultivo y añadir un volumen igual de PBS estéril 1x para lavar las células. </li><li> Añadir la cantidad necesaria paraformaldehído al 4% (PFA) para cubrir las células. Fijar las células durante 10 minutos a 4 ° C. </li><li> Retire PFA y lavar con explantes 1x PBST (PBS 1x con0,1% Tritón X-100) para permeabilizar las células. </li><li> Inmunotinción con anticuerpo deseado (por ejemplo, Alexa Fluor 488-faloidina; ZO-1, α-tubulina y actina de músculo liso α-) 7. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Es fundamental para el desarrollo de técnicas que facilitan el estudio del epicardio para atender a la creciente importancia del epicardio en el desarrollo cardiaco y la regeneración. El sistema de cultivo epicárdica plantea ventajas significativas para la investigación epicárdica. Una forma alternativa para aislar las células epicárdicas es utilizar la clasificación de células activadas por fluorescencia (FACS). Este método se basa en el uso de marcadores epicárdicas (o expresió...

Una gran cantidad de datos experimentales han puesto de manifiesto que el epicardio influye en los pasos críticos en el desarrollo cardíaco. Durante el desarrollo, el tabique transverso da lugar a un grupo de células mesoteliales conocidos como proepicardio 1-4. Las células de la proepicardio luego migran y el sobre el miocardio que forma el epicardio. Después de esto, un subconjunto de células epicárdicas someterse a aumento EMT dando a una población migratorio de las células derivadas del epicardio se (EPDCs) que posteriormente invaden el miocardio. linaje genético, así como el rastreo retroviral experimentos han demostrado que EPDCs diferenciarse en varios linajes, incluyendo células de músculo liso, fibroblastos, células endoteliales y cardiomiocitos (si los hay). Por lo tanto EPDCs contribuyen significativamente al desarrollo de la vasculatura coronaria y la arquitectura miocárdica 1,2,4-9. Además, el epicardio es esencial para el desarrollo de la capa compacta ventricular 10-12. Delanteroxample Gittenberger-de Groot et al. demostró que la inhibición de la excrecencia de la proepicardio conduce a una serie de defectos, tales como el miocardio delgada, looping deficiente del corazón y la formación de septum interventricular anormal y, como resultado, la letalidad embrionaria 13. factores paracrinos secretadas desde el epicardio embrionario modular la proliferación de los cardiomiocitos y la diferenciación. En consonancia con esto, la deleción-epicardio específica de las vías de señalización tales como el ácido retinoico (RA), factores de crecimiento de fibroblastos (FGFs) y Wnt / β-catenina resultó en el crecimiento del miocardio defectuoso y letalidad embrionaria 14-16. Aunque el epicardio se creía que era quiescente en corazones adultos, estudios recientes han demostrado que el programa de desarrollo se reactiva en el epicardio después de una lesión cardiaca 17,18. Tras la activación, las células se someten a una rápida proliferación y EMT que dan lugar a la formación de EPDC. Estas células exhiben el capacity de diferenciarse en fibroblastos y células musculares lisas pero no cardiomiocitos o células endoteliales 18. Además, los EPDCs secretan factores proangiogénicos que ayudan en la vascularización de la zona lesionada y así facilitar la mejora de la función cardíaca mediante la reducción del tamaño del infarto. Debido a estos resultados, el epicardio ha ganado interés en el estudio del desarrollo cardiovascular, la enfermedad y la regeneración. La tecnología transgénica ha revolucionado la investigación médica en el siglo 21. Con la ayuda de tecnologías transgénicas, los modelos de ratón enfermas que imitan la condición humana metabólicamente y fisiopatológico se han desarrollado con éxito. Sin embargo, el estudio del comportamiento de células epicárdica en estos mutantes ha sido un reto debido principalmente a la letalidad embrionaria temprana. Teniendo en cuenta el importante papel que juega el epicardio en el desarrollo y la regeneración cardiaca, hemos establecido un sistema de cultivo in vitro para ratón epicardiacélulas L. Este método permite que el cultivo a largo plazo de las células epicárdicas y facilita el estudio detallado de las dos propiedades importantes del epicardio: su capacidad de migrar y diferenciarse. Los ventrículos escindió del ratón podrían ser cultivadas en geles de colágeno que pueden ser utilizados para llevar a cabo los ensayos de migración. De haber sido cultivadas en una matriz 3D que reproduce la matriz extracelular rica en colágeno de la capa subepicárdica mejor recapitula la fisiología celular in vivo. Alternativamente, pueden ser cultivadas en portaobjetos de cámara con el fin de establecer una monocapa epicárdica que luego se puede utilizar para una variedad de aplicaciones aguas abajo. Esta monocapa se puede utilizar para teñir para proteínas de unión estrecha que proporcionará información sobre la capacidad del epicardio a someterse a EMT que es crucial para la migración. Además, los experimentos de diferenciación también pueden llevarse a cabo en estas células. Además, el perfil de expresión génica se puede analizar mediante la extracción de ARN de las células yla realización de la reacción en cadena de la polimerasa cuantitativa (qPCR). Por último, las monocapas también podrían ser tratados con agentes, seguido de un análisis molecular para probar posibles terapias. En conjunto, este sistema de cultivo epicárdica nos proporciona la oportunidad de visualizar y obtener datos molecular que promueve nuestro entendimiento sobre el desarrollo del epicardio. Otra característica deseable de este método es que es sencilla y no se requiere ninguna configuración compleja. En resumen, los embriones se recogieron a E11.5 E12.5 o después de lo cual se extirpó el corazón. Los ventrículos se cultivan a continuación en cualquiera de gel de colágeno o la cámara de diapositivas. Posteriormente, estas células se pueden utilizar para llevar a cabo experimentos de aguas abajo.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Life tech invitrogen&amp;nbsp;</td><td>11995065</td><td></td></tr><tr><td>Penicillin/streptomycin solution</td><td>Life tech invitrogen&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Life tech invitrogen&amp;nbsp;</td><td>10500064</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-5KG</td><td></td></tr><tr><td>Recombinant fibroblast growth factor 2 (FGF2)</td><td>PeproTech</td><td>450-33</td><td></td></tr><tr><td>Recombinant transforming growth factor beta 1 (TGF-&amp;beta;1)</td><td>PeproTech</td><td>100-21</td><td></td></tr><tr><td>ZO-1 antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>40-2200</td><td></td></tr><tr><td>&amp;alpha;-Tubulin antibody</td><td>Sigma</td><td>T 6074</td><td></td></tr><tr><td>&amp;alpha;-smooth muscle actin (SMA) antibody</td><td>Sigma</td><td>A 2547</td><td></td></tr><tr><td>Phalloidin antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>A12379</td><td></td></tr><tr><td>3D Collagen Culture kit&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ECM 675</td><td></td></tr><tr><td>8-well chamber slide</td><td>Fisher Scientific</td><td>NNU 154534-PK</td><td></td></tr><tr><td>Trizol reagent</td><td>Life Technologies</td><td>15596-018</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Life Technologies</td><td>4453536</td><td></td></tr><tr><td>Superscript First Strand Synthesis kit</td><td>Life Technologies</td><td>11904-018</td><td></td></tr></tbody></table>

in vitro culture of epicardial cells from mouse embryonic heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

El uso de este protocolo de cultivo, las células epicárdicas primarios se pueden aislar con alta pureza para aplicaciones posteriores. Las células cultivadas son capaces de someterse a EMT, migrar y diferenciarse al igual que las células epicárdicas hacen en vivo. Para determinar la pureza de nuestro cultivo de células del epicardio primaria, se analizaron los explantes epicárdicas generados a partir de Sem...

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In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Cultivo in vitro de células a partir de epicárdicos del corazón de embriones de ratón

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

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Research

JoVE Journal

Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.4K vistas.  Duke-NUS Graduate Medical School. The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. 

Video: In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Consecuencia del epicardio y Ensayo de Cultivo<em&gt; Ex Vivo</em&gt; Evaluación de la migración de células derivadas del epicardio

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Investigación

Biología del Desarrollo

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

El aislamiento y cultivo de células epicárdicas principales derivan de ejemplares de corazón humano adulto y Fetal

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In Vitro Generación de Células Progenitoras Cardíacas específicas de Mouse Field

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Transgénesis basada en electroporación de células cardíacas en corazones de ratón

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Autor destacado: Transgénesis de células cardíacas para el cribado rápido de genes

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

Un enfoque eficiente de transgénesis para la entrega de genes en el corazón embrionario de ratón

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Aislar las células melanocitos como primarios del corazón alfombrillas

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Biología

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Aislamiento de células endoteliales embrionarias ventriculares

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Cultivo in vitro de células a partir de epicárdicos del corazón de embriones de ratón

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Consecuencia del epicardio y Ensayo de Cultivo

El aislamiento y cultivo de células epicárdicas principales derivan de ejemplares de corazón humano adulto y Fetal

In Vitro Generación de Células Progenitoras Cardíacas específicas de Mouse Field

Un enfoque eficiente de transgénesis para la entrega de genes en el corazón embrionario de ratón

Un enfoque eficiente de transgénesis para la entrega de genes en el corazón embrionario de ratón

Un enfoque eficiente de transgénesis para la entrega de genes en el corazón embrionario de ratón

Aislar las células melanocitos como primarios del corazón alfombrillas

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

A Novel

Aislamiento de células endoteliales embrionarias ventriculares