Name: in vitro culture of epicardial cells from mouse embryonic heart
Uploaded: 2016-04-27T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

Este trabalho foi apoiado por fundos de Duke-NUS Graduate Medical School Singapura, Goh fundação e Singapura NRF comunhão (NRF-NRFF2016-01) para Manvendra K. Singh.

Todos os experimentos foram aprovados pelo Comitê Animal Care Institucional e Use a Duke-NUS Graduate Medical School. 1. Recuperar o Embryonic ventrículos <ol><li> Sacrificar um rato grávida cronometrado na fase embrionária desejado (E11.5 ou E12.5), utilizando uma câmara de eutanásia com fornecimento de gás dióxido de carbono ou outro método de eutanásia aprovado. </li><li> Posicione o mouse sobre as suas costas sobre uma mesa de dissecação. Desinfectar o abdômen da fêmea, com 70% de etanol. Levante a pele sobre a barriga e fazer uma pequena incisão (1-2 cm) com uma lâmina, em seguida, mantenha a pele com ambas as mãos e puxe para expor a parede abdominal. </li><li> Agora cortar a parede abdominal para expor o corno uterino. Use uma pinça estéreis para levantar o corno uterino e cortar cuidadosamente os tecidos de gordura e vasos sanguíneos ligados ao corno uterino. Corte no colo do útero para recuperar o útero. </li><li> Coloque o corno uterino em uma placa de petri contendo estéril frio 1x fosfato Buff<html> Ered Saline (PBS) e lave delicadamente. Colocar a placa de petri em gelo. </li><li> Utilize uma tesoura para cortar através da linha mediana da trompa uterina (oposto ao local onde a placenta está localizado) para expor os embriões que estão ainda dentro do saco vitelino e ligados a parede uterina através da placenta. </li><li> Utilizando uma pinça estéril, cortado do saco vitelino embrionário para liberar o embrião. </li><li> Coloque o embrião de uma nova placa de Petri contendo estéril frio 1x PBS. Decapitar o embrião e colocá-lo em suas costas. Cortado da parede torácica para expor o coração. </li><li> Use uma pinça estéreis para levantar o coração e cortar os vasos em torno dele para libertar o coração da parede torácica. Preste atenção especial para não danificar o epicárdio do coração com as ferramentas cirúrgicas. </li><li> Aparar a via de saída e ambos os átrios. Transferir o ventrículo a outro prato com 1x PBS frio. Coloque o prato no gelo. </li><li> Repita os passos de 1,6-1,9 até que todos os ventrículos são recuperadas. </li></ol>le &quot;&gt; 2. Cultura Explant Epicárdica Nota: Cultura esses explantes do epicárdio, quer em lâminas de câmara de vidro ou em gel de colágeno para aplicações a jusante. <ol><li> Slides câmara de vidro <ol><li> Para preparar o meio de cultura, adicionar 10% de soro fetal de bovino (FBS), 1% de penicilina / estreptomicina e 2 ng / ml de factor de crescimento de fibroblastos recombinante 2 (FGF2) para meio de Eagle modificado por Dulbecco (DMEM). Executar todas as etapas da capela de fluxo laminar. </li><li> Adicionar 500 ul de meio de cultura a cada poço de uma câmara de 8 poços. </li><li> Colocar cada ventrículo excisado no centro de um poço. Orientar o ventrículo para manter o lado dissecados para baixo sobre a superfície do fundo do poço. </li><li> Suavemente colocar a placa num banho a 37 ° C, 5% de CO 2 incubadora de cultura de células. </li><li> Manter a cultura com uma perturbação mínima para permitir que os explantes a aderir. A formação da monocamada epicárdio pode ser observada depois de 48-72 horas. </li><li> Para a diferenciação de epicardcélulas ial em células musculares lisas, a cultura das células do epicárdio monocamada em meio de diferenciação por mais 6 dias. Para preparar os meios de diferenciação, adicionam-se 10% de FBS, 1% de penicilina / estreptomicina e 50 ng / ml de factor de crescimento de transformação recombinante beta 1 (TGF-β1) para DMEM. </li><li> Alterar o meio de cultura em dias alternados. </li></ol></li><li> colágeno Gel <ol><li> Prepara-se o gel de colagénio numa placa de 96 poços utilizando um kit de cultura 3D colagénio comercial de acordo com o protocolo do fabricante. </li><li> Para preparar o volume desejado de gel de colagénio, misturar a quantidade apropriada de solução de colagénio com 5x de DMEM e pipeta cima e para baixo. A solução deve ficar amarelo. </li><li> Adicionar um volume correspondente de solução de neutralização e misture bem imediatamente pipetando cima e para baixo. A solução vai virar rosa. </li><li> Pipetar 100 ul desta mistura em cada poço de uma placa de 96 poços. Colocar a placa num banho a 37 ° C, 5% de CO2 durante 30-60 min para albaixo o gel para polimerizar. </li><li> Adicionar 200 ul de meio de cultura descritas acima para cada poço. </li><li> Colocar cada ventrículo excisado no centro de um poço. Orient o ventrículo para manter o lado dissecados para baixo sobre o gel de colágeno (ápice do ventrículo deve enfrentar o experimentador). Suavemente colocar a placa de volta na incubadora de cultura de células. </li><li> Após 3 dias, remover os ventrículos. Colocar a placa de volta na incubadora de cultura de células durante mais 2 dias. </li><li> Alterar o meio de cultura em dias alternados. </li></ol></li></ol> 3. Fixação e Visualização Nota: as células epicárdicas pode ser visualizado em ambos lâminas de câmara de vidro ou de gel de colagénio. <ol><li> Aspirar o meio de cultura e adicionar um volume igual de PBS estéril 1x para lavar as células. </li><li> Adicionar apenas o suficiente paraformaldeído a 4% (PFA) para cobrir as células. Fixar as células durante 10 min a 4 ° C. </li><li> Remover e lavar PFA explantes com 1x PBST (PBS 1x com0,1% de Triton X-100) para permeabilizar as células. </li><li> Imunocoloração com anticorpo desejado (por exemplo, Alexa Fluor 488-faloidina; ZO-1, α-tubulina e actina de músculo liso-α) 7. </li></ol>

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

É crucial para desenvolver técnicas que facilitam o estudo do epicárdio para atender à crescente importância do epicárdio no desenvolvimento cardíaco e regeneração. O sistema de cultura do epicárdio representa vantagens significativas para a investigação do epicárdio. Um modo alternativo para isolar as células do epicárdio é a utilização de triagem de células activadas por fluorescência (FACS). Este método baseia-se na utilização de marcadores do epicárdio (ou express...

A riqueza de dados experimentais têm mostrado que o epicárdio influencia passos críticos no desenvolvimento cardíaco. Durante o desenvolvimento, o septo transverso dá origem a um grupo de células mesoteliais conhecidos como o proepicardium 1-4. As células do proepicardium seguida, migrar e envelope do miocárdio formando o epicárdio. Depois disso, um subconjunto de células do epicárdio sofrer EMT dando origem a uma população de células derivadas migratória-epicárdio (EPDCs) que invadem posteriormente o miocárdio. Genética retroviral, bem como a linhagem rastreio experimentos demonstraram que EPDCs diferenciar em linhagens diferentes, incluindo células musculares lisas, fibroblastos, células endoteliais e cardiomiócitos (se qualquer). Portanto EPDCs contribuir significativamente para o desenvolvimento da vasculatura coronária e miocárdica arquitectura 1,2,4-9. Além disso, o epicárdio é essencial para o desenvolvimento da camada compacta do ventrículo 10-12. para example Gittenberger-Groot et ai. demonstraram que a inibição do crescimento do proepicardium leva a uma variedade de defeitos, tais como uma fina miocárdio, looping deficiente do coração e septo interventricular formação anormal e, como resultado, a letalidade embrionária 13. factores parácrinos secretadas a partir do epicárdio embrionário modular a proliferação e diferenciação de cardiomiócitos. Consistente com isto, supressão específica do epicárdio de vias de sinalização, tais como o ácido retinóico (RA), factores de crescimento de fibroblastos (FGF) e Wnt / β-catenina resultou no crescimento do miocárdio com defeito e letalidade embrionária 14-16. Embora o epicárdio se acreditava ser de repouso em corações adultos, estudos recentes têm mostrado que o programa de desenvolvimento é reativado no epicárdio após lesão cardíaca 17,18. Após a activação, as células sofrem uma rápida proliferação e EMT que resultam na formação EPDC. Estas células exibem a capacitY para se diferenciarem em fibroblastos e células do músculo liso, mas não cardiomiócitos e células endoteliais 18. Além disso, os pró-angiogénico EPDCs segregam factores que ajuda na vascularização da área ferida e, assim, facilitar a melhoria da função cardíaca ao reduzir o tamanho do enfarte. Devido a estes resultados, o epicárdio ganhou interesse no estudo do desenvolvimento cardiovascular, doença e regeneração. tecnologia transgênica tem revolucionado a investigação médica no século 21. Com o auxílio de tecnologias transgénicos, modelos de ratos doentes que mimetizam a condição humana metabolicamente e fisiopatologicamente ter sido desenvolvida com sucesso. No entanto, o estudo do comportamento das células do epicárdio nestes mutantes tem sido um desafio devido principalmente a letalidade embrionária precoce. Considerando o papel significativo que o epicárdio desempenha no desenvolvimento cardíaco e regeneração, nós estabelecemos um sistema de cultura in vitro para epicardia ratocélulas L. Este método permite que a cultura de longo prazo das células do epicárdio e facilita o estudo detalhado das duas propriedades importantes do epicárdio: a sua capacidade para migrar e diferenciar-se. Os ventrículos cortados do rato poderia ser cultivados em géis de colagénio que podem ser utilizados para conduzir ensaios de migração. Sendo cultivadas em uma matriz 3D que replica a matriz extracelular rica em colágeno da camada subepicárdica recapitula a na fisiologia da célula vivo melhor. Alternativamente, eles podem ser cultivadas em lâminas de câmara, a fim de estabelecer uma monocamada epicárdico que pode então ser utilizado para uma variedade de aplicações a jusante. Esta monocamada pode ser utilizado para corar para proteínas de junções apertadas, que irá proporcionar conhecimentos sobre a capacidade do epicárdio se submeter EMT que é crucial para a migração. Além disso, as experiências de diferenciação pode também ser levada a cabo sobre estas células. Além disso, o perfil de expressão do gene pode ser analisado através da extracção de ARN a partir das células erealização da reacção em cadeia da polimerase quantitativa (qPCR). Por último, as monocamadas também poderiam ser tratados com agentes seguido por uma análise molecular para testar potenciais agentes terapêuticos. Juntos, este sistema de cultura epicárdica nos proporciona a oportunidade de visualizar e reunir dados molecular que possibilita a compreensão sobre o desenvolvimento do epicárdio. Outra característica desejável deste método é que é simples e não é necessária configuração elaborada. Em resumo, os embriões são colhidos em E11.5 E12.5 ou após o que o coração é excisado. Os ventrículos são então cultivadas em qualquer um gel de colagénio ou lâminas de câmara. Subsequentemente, estas células podem ser usadas para conduzir experiências jusante.

<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.

Usando este protocolo cultura, as células do epicárdio primárias podem ser isolados com elevado grau de pureza para aplicações a jusante. As células cultivadas são capazes de sofrer EMT, migrar e diferenciar células do epicárdio, assim como faz in vivo. Para determinar a pureza da nossa cultura de células do epicárdio primário, analisamos os explantes do epicárdio gerados a partir Sema3d <sup...

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In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Cultura in vitro de células epicárdicas De Coração embrionárias de camundongos

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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JoVE Journal

Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.3K visualizações.  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. 

Vídeo: 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.

Epicárdio Conseqüência Cultura Ensaio e<em&gt; Ex Vivo</em&gt; Avaliação da migração celular Epicárdica derivado

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

Biologia do Desenvolvimento

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.

O isolamento e a cultura de células primárias de Epicárdica derivado de espécimes de coração adulto e Fetal humano

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 geração de células progenitoras cardíacas específicas do coração do rato

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

Transgênese baseada em eletroporação de células cardíacas em corações de camundongos

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 em destaque: Transgênese de células cardíacas para triagem rápida 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.

Uma abordagem eficiente de transgênese para entrega de genes no coração embrionário de camundongos

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;

Isolar células de melanócito-like primárias do coração mouse

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 ...

Biologia

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.

Isolamento e Cultura de Neonatais rato Cardiomyocytes

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 Cultura para o Rato coração embrionário

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.

Isolamento de Células Embrionárias ventriculares endoteliais

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

Cultura in vitro de células epicárdicas De Coração embrionárias de camundongos

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