Os autores graças a Nadine Woitasky e Peter Volk para assistência técnica. Além disso, os autores graças a Sra. Claudia Lorenz (médico escritor, ACCEDIS) por sua ajuda na preparação do manuscrito. Este manuscrito foi apoiado financeiramente pelo DFG (Schlu 324/7-1).

a investigação é conduzida de acordo com o guia para o cuidado e o uso de animais de laboratório publicado por nos National Institute of Health (NIH publicação n º 85-23, revista em 1996). Em geral, ratos wistar machos com 3 a 4 meses de idade e com um peso médio de 250-350 g são utilizados para este protocolo. Um coração de rato é suficiente para 20 pratos de cultura (1 mL por prato; diâmetro interno: 35 mm) com uma densidade celular aproximada de 1,5 x 10 4 células/1000 mm 2.
 1. preparação de meios e reagentes <ol><li>meio de creatina-carnitina-taurina (meio CCT) Nota: CCT medium é um meio complexo, baseado no meio 199 com a adição de creatina, carnitina e taurina.
	<ol><li>Preparar 1 L de meio 199: Adicione 3,6 g Hepes e misture por 1h. Em seguida, adicione creatina 655,5 mg (5 mM), carnitina 395,4 mg (2 mM) e taurina 625,5 mg (5 mM). Carnitina e taurina alteram o pH a &#60; 7. A fim de inibir o crescimento de células, por exemplo, células endoteliais ou fibroblastos contaminante, adicione 10 µM citosina β-D-arabinofuranoside ao meio. Ajuste o pH com NaOH (2 mM) ao filtro de 7,4 e estéril o meio. Armazenar o meio CCT em 4 ° C.</li>
	</ol></li> <li>Médio de Powell <ol><li>por meio de 1 L Powell, dissolver 6,43 g NaCl (110 mM) com 0,19 g KCl (2,5 mM), 0,16 g KH 2 PO 4 mM (1,2), 0,3 g de MgSO 4 7 H 2 O (1,2 mM), 5,96 g Hepes (25 mM) e 1,98 g D (+ )-Monohydrate da glicose (10 mM) no Aqua estéril. Ajuste o pH com NaOH (M 2) 7.4 e estéril filtro o meio. Médio de Powell loja a 4 ° C.</li>
	</ol></li> <li>De cloreto de cálcio (CaCl 2) <ol><li>preparar uma solução de 100 mM CaCl 2 (50 mL) e preparar alíquotas contendo 500 µ l de CaCl 2. Congelar as alíquotas a -20 ° C.</li>
	</ol></li> <li>Preparação do meio de cultura <ol><li>preparar três meios de cultura celular: Pré-chapeamento médio, chapeamento médio e médio de lavagem. Use meio de CCT como a base para todas as três mídias (tabela 1). Calcule a média CCT com 1ml por prato de cultura. Portanto, prepare-se 20 mL de meio de CCT para 20 pratos de cultura (diâmetro interno: 35mm).</li>
		<li>Célula cultura placas: revestir cada placa de cultura de células (diâmetro interno: 35mm) com 1 mL Pré-chapeamento médio. Armazenar as placas revestidas a 37 ° C durante a noite ou pelo menos 2 h antes de usar.</li>
	</ol></li></ol> 2. Isolamento de adulto Cardiomyocytes <ol><li>sistema de preparação de Langendorff perfusão <ol><li>calor médio de chapeamento e lavagem médio a 37 ° C. um tubo de 500 µ l de CaCl 2 de descongelar e pesar em 25 mg de colagenase.</li>
		<li>Irrigue o sistema de perfusão de Langendorff com aqua estéril, depois deixe médio Powell circular o sistema 5 min.</li>
		<li>Encher o sistema de perfusão de Langendorff com 80 mL de meio de Powell, sem qualquer ar bolhas e gás médio com 95% de oxigênio.</li>
		<li>Preparar um tubo (50 mL) com 40 mL de meio de Powell, aquecê-lo a 37 ° C e gás-com 95% de oxigênio.</li>
		<li>Preparar um segmento de cerca de 25 cm de comprimento para prender o coração removido para a cânula.</li>
		<li>Desengordurar uma lâmina de barbear com álcool (70% em volume) e aperte-o para o helicóptero. Fixar um disco de plástico no helicóptero.</li>
	</ol></li> <li>Extração de coração <ol><li>anestesiar um rato wistar machos com 4 a 5% de isoflurano e sacrificá-lo com deslocamento cervical. Abrir o abdômen para trás o arco costal com uma tesoura abdominal e, com a mesma tesoura, corte através do diafragma para abrir a cavidade torácica.</li>
		<li>Remover o coração, junto com o pulmão e o timo, cortando acima o Timo altamente craniano na cavidade torácica. Transferir o material para solução salina gelada imediatamente.</li>
		<li>Remover o pulmão e Timo do coração com uma tesoura de dissecação (grande) e pela fixação do material com cápsula fórceps, transferir este último para uma nova solução salina.</li>
	</ol></li> <li>Isolamento <ol><li>remover o excesso de tecido, como resíduos de timo, traqueia, gordura e tecido conjuntivo do coração usando fórceps da cápsula e uma tesoura de dissecação (grande ou pequena). Descubra a aorta e é cortar com uma tesoura de dissecação (grande ou pequena) entre o primeiro e segundo branquial arq</li>
		<li>Começar a pingar do sistema de perfusão de Langendorff. Coloque o coração sobre a cânula do sistema de perfusão de Langendorff e fixá-lo primeiro com uma pinça de crocodilo e mais tarde com o segmento preparado. Enxágue o coração até que ele está livre do sangue.</li>
		<li>Dissolver 25 mg colagenase em 5-6 mL de meio de Powell quente e adicionar 12,5 µ l CaCl 2 (30 µM).</li>
		<li>Fechar a circulação movendo um funil de vidro, que é conectado com o sistema de perfusão de Langendorff, sobre o coração de gotejamento e adicionar a colagenase resolvido para o sistema de perfusão. Iniciar a perfusão para 25 min com uma velocidade de queda de 1 gota por segundo. 
		Nota: Durante a perfusão, o coração vai inchar e obter uma aparência cerosa.</li>
		<li>Parar a perfusão após 25 min e retirar o coração do sistema de perfusão de Langendorff. Remover a aorta, átrios e tecido conjuntivo do coração e abrir os ventrículos direito e esquerdos.</li>
		<li>Corta o coração duas vezes em um ângulo de 90° (largura de corte: 0,7 mm; velocidade: 0,15 cm/s). Repita este processo manualmente com dois bisturis para 10 s cada lado.</li>
		<li>Transferir 12 mL do meio de perfusão para um novo tubo (50 mL). Despeje o chorume de célula este médio e digerir células por mais cinco minutos a 37 ° C. Mix a solução cada minuto.</li>
		<li>Filtrar a solução com o coração digerido através de uma malha de nylon (200 µm) para um tubo novo (50 mL).</li>
		<li>Centrifugar a solução filtrada em 29 x g, durante 3 min. descartar o sobrenadante e adicionar 6 mL quente Powell de meio incluindo 12,5 µ l CaCl 2 (250 µM) para o centrifugado. Resuspenda o pellet através de movimentos de agitação suaves. Centrifugar novamente a 29 x g por 2 min. descartar o sobrenadante e adicionar 6 mL quente médio Powell substituído com 25 µ l CaCl 2 (500 µM). Dissolver o centrifugado através de movimentos suaves de agitação e adicione 12 mL quente Powell médio incluindo 120 µ l CaCl 2 (1 mM). Centrífuga para uma terceira vez em 16 x g por 1 min. Novamente, remover o sobrenadante.</li>
		<li>Misturar o centrifugado com o médio pré-aquecido chapeamento.</li>
		<li>Remover Pré-chapeamento médio de placas de cultura. Transferi 1 mL do chapeamento médio, incluindo o cardiomyocytes isolada, para cada placa de cultura. Incube fresco cardiomyocytes isolado a 37 ° C por 1 h.</li>
		<li>Remover o meio de chapeamento de placas de cultura. Adicione 1 mL de lavagem média para cada placa de cultura e armazenar as placas a 37 ° C até seis dias sem alterar o meio.</li>
		<li>Para investigar a influência de diferentes produtos químicos e tratamentos na ARVC, primeiro atualizar o chapeamento médio médio de lavagem e posteriormente adicionando produtos químicos diferentes. 
		Nota: Avaliação com microscopia de luz: com preparação cada célula, 150 a 300 cardiomyocytes deve ser monitorados por dia por microscopia de luz. Subdividir a cardiomyocytes todos contados em grupos de acordo com a sua aparência (por exemplo, &#34; em forma de haste &#34;, &#34; redonda para baixo &#34;, &#34; espalhando &#34;, e &#34; aparência incomum &#34;). A categoria &#34; espalhando &#34; inclui todos cardiomyocytes com pseudópodos-como estruturas. &#34; Aparência incomum &#34; inclui todos ARVC com uma superfície irregular e não detectável intacta da membrana celular.</li>
	</ol></li></ol> 3. Exemplo de Experimentos <ol><li>fluorescência/imunofluorescência coloração dos adultos cardiomyocytes <ol><li>análise morfológicas e estruturais conversões da ARVC durante o cultivo por microscopia confocal do laser. Use a faloidina-TRITC para investigar estruturas F-Actina em &#34; em forma de haste &#34;, &#34; redonda para baixo &#34;, e &#34; espalhando &#34; ARVC. Realizar a coloração de acordo com o fabricante &#39; protocolo s. Um exemplo para a faloidina-TRITC coloração é dada em referência Nippert et al 11. com coloração de fluorescência/imunofluorescência, diferenças no de - e re - differentiation do aparato contrátil em cardiomyocytes adultos cultivadas entre tratamentos experimentais (por exemplo, com Swiprosin-1, ionomycin) pode ser investigado.</li>
	</ol></li> <li>RT-PCR quantitativo em tempo real (qRT-PCR) <ol><li>executar qRT-PCR para investigar alterações na expressão do mRNA de genes diferentes (por exemplo, OSM, Swiprosin-1, β-MHC) durante o cultivo da ARVC. Para o tamanho de amostra suficiente, use pratos de cultura maiores (diâmetro interno: 60 mm) com volume de 2 mL. ARVC de cinco pratos de cultura produz uma amostra. Realizar isolamento do mRNA e a transformação do cDNA de acordo com o fabricante &#39; protocolo de s.</li>
	</ol></li> <li>Immunoblot técnicas <ol><li>realizar borrões ocidentais para investigar alterações na expressão de proteínas (por exemplo, para Swiprosin-1) durante o cultivo da ARVC. Use um prato de cultura (1 mL) por exemplo.</li>
	</ol>
	</li>
</ol>

<ol>
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Os resultados mostrados são parte da tese de doutorado de Franziska Nippert.

O comportamento do adulto cardiomyocytes na vivo é influenciado por muitas interações com outras células (por exemplo, os neurônios, células endoteliais, fibroblastos, células inflamatórias) e o sincício elétrico que formam1. Portanto, estudar a adaptação de estresse de adultos cardiomyocytes exclusivamente requer o isolamento e cultivo da ARVC. Os principais efeitos de isolar e cultivar ARVC são: 1) desconectá-los de matriz extracelular e contatos do celular; 2) de...

Cardiomyocytes adulto na vivo funciona como um sincício elétrico baseado em contatos de celular entre myocytes. Além disso, eles são influenciados por células adjacentes como cardíacos fibroblastos, células endoteliais, neurônios e células inflamatórias1. Para estudar a capacidade dos cardiomyocytes para adaptar sua organização intracelular para alterou as condições de carga, como visto durante a hipertrofia cardíaca, que é um passo inicial levando a insuficiência cardíaca, o isolamento e cultivo de adulto rato ventricular cardiomyocytes (ARVC) é necessário2,3,4. Historicamente, cardiomyocytes primeiro foram isoladas de garota embrionárias corações5,6. Alguns anos mais tarde, o primeiro isolamento de cardiomyocytes terminalmente diferenciada foi descrito por meio de depleção de cálcio7. No entanto, esses adultos cardiomyocytes não eram tolerantes de cálcio e, portanto, não podiam ser utilizados para os ensaios funcionais. Finalmente, em 1976, um novo protocolo habilitado Powell e torcer para investigar adulto cardiomyocytes ventricular sob condições fisiológicas8. Como primeiro passo, isolaram cardiomyocytes adulto sob concentrações baixas de cálcio e cálcio posteriormente aumentado para concentrações fisiológicas em um processo gradual. Hoje, a maioria dos protocolos para isolamento e cultivo de adultos cardiomyocytes trabalharcom com este protocolo de cálcio e usam colagenase para a digestão enzimática da célula-célula densa contatos1.
Para um cultivo bem sucedido, é necessário soro fetal bezerro (FCS) ou oncostatin M (OSM). ARVC executar um de - e re - differentiation extensas alterações estruturais, incluindo o sarcômero desmontagem e reforma9,10,11,12. Este processo é acompanhado por uma re-expressão de genes fetais-tipo, como β-miosina de cadeia pesada (MHC-β), como conhecido de hipertrofia e uma formação de pseudópodos-como estruturas, também chamado de espalhamento4,11, 13. Além disso, swiprosin-1 (EFHD2), uma proteína recém identificada, desempenha um papel importante no processo de re-diferenciação de cultivada ARVC11. Como resultado, ARVC na cultura transformar-se em células generalizadas, polimórficas, que mostram espontaneamente contrações após duas a três semanas em cultura2,4,14.
Descobertas recentes têm revelado que cardíaca differentiation re e de como ocorre em condições de cultura imita possui visto na vivo durante cardíaca remodelação10,15. Remodelação cardíaca é um processo chave durante doenças cardíacas16. Como doenças cardíacas ainda são a principal causa de morte nas sociedades industrializadas, uma melhor compreensão da biologia da cardiomyocytes adulto é importante (quem; 2015). Isolamento e cultivo da ARVC podem ajudar a desenvolver novas estratégias e medicamentos para o tratamento de doenças cardíacas. Com este manuscrito, é fornecido um protocolo para o isolamento e cultivo da ARVC. Além disso, algumas partes críticas deste método são destacadas na seção de discussão.

<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

Em um coração intacto, células adjacentes influenciam cardiomyocytes adulto. Com o método de isolamento e cultivo de adultos cardiomyocytes, uma investigação precisa do comportamento dessas células sob ambientes e tratamentos específicos é possível. Este manuscrito apresenta um protocolo para a bem sucedido isolamento e cultivo de rato adulto cardiomyocytes ventricular (ARVC).
O rato é sacrificado por deslocamento cervical sob anestesia profunda. Então, é extraído o coração e a aorta é descoberta. Posteriormente, a perfusão do sistema de perfusão de Langendorff com depleção de cálcio e tratamento de colagenase é executada. Depois, tecido ventricular Obtém picado recirculado e filtrado, seguido por três etapas de centrifugação com adição gradual de CaCl2 até que seja alcançada a concentração de cálcio fisiológica. ARVC são banhados em pratos de cultura de células. Após atualizar o meio de cultura celular, ARVC pode ser cultivada por até seis dias sem alterar o meio de cultura contendo soro. Isolamento da ARVC é um processo sensível de cálcio. Pequenas alterações da concentração de cálcio intracelular causar uma diminuição na qualidade e viabilidade das células isoladas.
Recentemente isolado ARVC são em forma de haste. Nos primeiros dias de cultivo, eles perdem os bacilares morfologia e forma pseudópodos estruturas (espalhar). Durante esta formação morfológica ARVC inicialmente degradar seus elementos contráteis, seguidos por uma reforma através de fibras de estresse de actina e sarcomerogenesis de novo . Após uma semana de cultivo, a maioria ARVC mostrar uma aparência generalizada com uma faixa transversal claramente detectável. Este processo é sensível à concentração de cálcio intracelular, como tratamento com ionomycin atenua a propagação. Marcadores chaves neste processo de - e re - differentiation são cadeia pesada de miosina-β (β-MHC), oncostatin M (OSM) e swiprosin-1 (EFHD2). Estudos recentes têm sugerido que cardíaca re e de differentiation ocorrendo em condições de cultura imita características visto na vivo durante a remodelação cardíaca. Portanto, isolamento e cultivo da ARVC desempenham um papel chave na compreensão da biologia dos cardiomyocytes.

Adulto cardiomyocytes na cultura: A Figura 1 mostra uma visão geral dos cardiomyocytes adultos recentemente isolado 2 h após a última lavagem. Aproximadamente 75% de todos os cardiomyocytes tinha uma morfologia de forma cilíndrica. Os restantes 25% mostrou uma aparência incomum com uma morfologia redonda e não detectável membrana celular intacta (Figura 1). No final do cultivo (dia 6), até 15% de todos os...

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

Isolation and Cultivation of Adult Rat Cardiomyocytes

Aqui, apresentamos um protocolo para o isolamento e cultivo de rato adulto cardiomyocytes ventricular (ARVC). ARVC isolado pode ser usado para o cultivo de curto e longo prazo. O isolamento e cultivo da ARVC podem desempenhar um papel chave no desenvolvimento de novos esquemas de tratamento para doenças cardíacas.

Isolamento e cultivo de rato adulto Cardiomyocytes

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

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Research

JoVE Journal

Medicine

15.5K visualizações.  Justus Liebig University Giessen. Aqui, apresentamos um protocolo para o isolamento e cultivo de rato adulto cardiomyocytes ventricular (ARVC). ARVC isolado pode ser usado para o cultivo de curto e longo prazo. O isolamento e cultivo da ARVC podem desempenhar um papel chave no desenvolvimento de novos esquemas de tratamento para doenças cardíacas.

Vídeo: 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.

O isolamento e criopreservação de Neonatais Rato Cardiomyocytes

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

Biologia do Desenvolvimento

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.

Cultura primária de Miócitos Coração Adulto Rat

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

Biologia

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.

Isolamento e manipulação genética de adultos miócitos cardíacos for Imaging 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.

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 ...

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.

Isolamento, cultura e caracterização funcional de rato 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.

Isolamento dos Cardiomyocytes Atrial de um modelo do rato de metabólicas relacionadas com síndrome de insuficiência cardíaca com fração de ejeção 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.

Isolamento e Cultura de rato adulto Cardiomyocytes de sinalização celular e<em&gt; In vitro</em&gt; A 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.

Isolamento, cultura e Transdução de rato adulto Cardiomyocytes

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.

Um Método Simples e Eficaz para Isolar Consistentemente Cardiomiócitos de Camundongos

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.

Isolamento Simultâneo de Cardiomiócitos de Alta Qualidade, Células Endoteliais e Fibroblastos de um Coração de Rato 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 ...