Name: culturing mouse cardiac valves in the miniature tissue culture system
Uploaded: 2015-10-19T14:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

Este protocolo sigue las directrices LUMC del comité de ética de la investigación animal. 1. Preparación de instrumentos, medio de cultivo, y MTCS Nota: Realice todas las preparaciones en la campana de flujo laminar. La cámara de perfusión MTCS, trampa de burbujas y el soporte se describen en Lieber et al., 2010 11. <ol><li> Desinfectar los fórceps y micro tijeras con etanol al 70%. Preparar una jeringa de 5 ml con una aguja 21G con NaCl estéril de Tyrode Buffer (130 mM, 5,4 mM KCl, mM Hepes 6,0, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, mM glucosa 20, 1,5 mM CaCl 2 • 2H 2 O, pH = 7,2). Preparar una jeringa de 5 ml con una aguja 21G con una solución de KCl estéril (100 mM). </li><li> Preparar 65 ml de medio por DMEM: corazón suplementado con 10% suero de ternero fetal, de antibiótico / antimicótico (A / A; 100 unidades de penicilina, 100 g de estreptomicina y 0,25 ug de anfotericina B / ml), insulina-Transferrin-El selenio (ITS; 10 mg / ml de insulina, 5,5 mg / ml de transferrina, 6,7 ng / ml selenita de sodio) y esterilizar filtro. </li><li> Esterilizar la cámara de perfusión y la trampa de burbujas mediante pulverización con etanol al 70% y secar con una toalla de papel. Montar el MTCS (Figura 1D), sin embargo, inicialmente sin la cámara de perfusión. Coloque la trampa de burbujas en el estrado. Utilice una bomba de rodillo peristáltico con cabezales de bomba fácil de carga. <ol><li> Uso de tubo de silicona hacer conexiones entre el depósito (tubo de 50 ml) y la bomba, la bomba y la trampa de burbujas, la trampa de burbujas y el depósito (véase la Figura 1D). Hacer el tubo dentro de la incubadora tiempo suficiente para permitir medio alcance el equilibrio de gas con el gas en la incubadora a través de la tubería de gas permeable de silicio (1 m) y el tubo fuera de la incubadora tan corto como sea posible. </li></ol></li><li> Llene el tubo de 50 ml con etanol al 70%, encienda la bomba (velocidad de flujo alrededor de 1 ml / min para permitir la circulación adecuada) y dejar que el cir etanolcular durante 30 min para esterilizar todos los tubos. Separar el etanol mediante el vaciado del depósito y bombear el etanol a partir del sistema. </li><li> Llene el tubo de 50 ml con agua destilada estéril, encender la bomba y deje circular el agua durante 30 minutos para deshacerse de etanol restante. Eliminar el agua vaciando el depósito y bombear el agua desde el sistema. </li><li> Llene el tubo de 50 ml con 45 ml de medio, poner el pie en la incubadora. Encienda el (velocidad de flujo alrededor de 1 ml / min) la bomba y circule el medio para llenar toda la tubería, retire todas las burbujas de aire y permitir que el medio para adaptarse a la composición del gas en la incubadora durante al menos 1 hora. Mantener la incubadora a condiciones de cultivo de tejidos estándar (5% de CO 2 y 37 ° C). Asegúrese de que no hay burbujas en el tubo. </li></ol> 2. Aislamiento de ratón del corazón <ol><li> Inyectar el ratón con 500 unidades de heparina. Esto evitará que la sangre se coagule y permite la eliminación de blood desde el corazón. Después de 10 min, anestesiar el ratón en una cámara de inducción con 4% de isoflurano usando un vaporizador de precisión. La anestesia es suficiente cuando el ratón no responde a la pizca dedo del pie. </li><li> Transferir el ratón para un tablero de disección y mantener la anestesia por medio de una mascarilla conectada a un circuito coaxial. Desinfectar la piel del ratón con un 70% de alcohol. Usando tijeras abiertas de la cavidad abdominal para exponer la vena cava y el diafragma. </li><li> Para exponer el corazón, hacer incisiones laterales a partir de la última a las primeras costillas y reflejan la caja torácica sobre la cabeza de ratón. Detener la anestesia como el ratón no está respirando más. Observar los latidos del corazón. </li><li> Insertar la aguja 21G unida a una jeringa 5 ml que contiene tampón estéril de Tyrode en la vena cava inferior desde el abdominal en la cavidad torácica (a través de diafragma). Asegúrese de que la sangre pueda salir de la vena cava caudal de la inserción de la aguja. Perfundir ªBuffer de correo Tyrode con suavidad y con una presión constante en la vena cava hasta que el corazón pierde en parte su color rojo. Nota: El corazón permanece superando lo que permite la extracción de la sangre desde el corazón. </li><li> Vuelva a colocar la aguja con la aguja 21G unida a una jeringa de 5 ml que contiene la solución KCl estéril. Perfundir suavemente la solución de KCl en la vena cava hasta que el corazón deja de latir y retire la aguja. Nota: La solución de KCl conserva el corazón en la fase diastólica relajado, lo que permitirá la inserción más fácil de las agujas de perfusión y la perfusión del sistema coronario. </li><li> Levante ligeramente el corazón utilizando pinzas curvas y con unas tijeras diseccionar el corazón libre de los tejidos circundantes, pero dejan las arterias y las venas proximales al corazón intacto y unido al corazón. Transferir el corazón a un tubo de 15 ml que contiene PBS enfriado con hielo suplementado con antibiótico / antimicótico (A / A). Guarde el corazón helado de PBS durante un máximo de 3 horas. </li></ol> 3. Cannulation de los corazones del ratón en la cámara de perfusión <ol><li> Realizar todos los procedimientos en campana de flujo laminar. Transferir el corazón aislado del tubo de 15 ml a una placa de 10 cm Petri y añadir PBS suplementado con A / A. </li><li> Bajo un microscopio de disección, utilice la tijera micro y fórceps para eliminar todo el tejido no cardiaco pero preservar la aorta ascendente al menos a la bifurcación con la arteria braquiocefálica y las venas pulmonares, 2 mm proximal al corazón. Cortar la punta de la punta del corazón para crear el acceso al lumen del ventrículo izquierdo (típicamente 2 mm). Coloque la cámara de perfusión en el campana de flujo bajo el microscopio de disección. </li><li> Adjuntar una jeringa de 5 ml con medio a la aguja de inserción usando el tubo de silicona. Llenar la cámara de perfusión con 20 ml de medio y poner el corazón en la etapa de rotación entre las 2 agujas romas en la cámara de perfusión (Figura 1). Ajuste la altura de la etapa de rotación por lo que el corazón se coloca delante de laagujas. </li></ol> 4. La ligadura para el cultivo de la válvula mitral (ver Figura 1) <ol><li> Ligar la aorta con sutura (seda 7.0). Ligar la orejuela auricular izquierda con sutura. Insertar la aguja (nr. 1 en la Figura 1) a través de la vena pulmonar hacia la aurícula izquierda y se liga con sutura proximal de su entrada en la aurícula izquierda. Asegúrese de que la aguja no está demasiado lejos en la aurícula izquierda ya que esto podría dañar la válvula mitral. </li><li> Insertar la aguja (nr.2 en la Figura 1) con movimiento lineal en el ventrículo izquierdo. Transferir el medio procedente de la cámara de perfusión a un tubo de 50 ml. Sellar la aguja al miocardio con pegamento biocompatible. </li><li> Después de que el pegamento se haya secado, inyecte cuidadosamente medio en el corazón mediante la conexión de una jeringa llena de mediano a la aguja nr.1 y compruebe si hay alguna fuga. Asegúrese de que las salidas de medianas de nr.2 aguja y la última sangre restante se perfunde fuera del corazón. </li></ol> 5. Ligación para el cultivo de la válvula aórtica (ver Figura 1) <ol><li> Insertar la aguja (Nr.1 en la Figura 1) en la aorta y se liga con sutura. Asegúrese de que la aguja no está demasiado lejos hacia la aorta ya que esto podría dañar la válvula aórtica. Insertar la aguja (nr.2 en la Figura 1) con movimiento lineal en el ventrículo izquierdo. Transferir el medio procedente de la cámara de perfusión a un tubo de 50 ml. Sellar la aguja al miocardio con pegamento biocompatible. </li><li> Después de que el pegamento se haya secado, inyecte cuidadosamente medio hasta el corazón mediante la conexión de una jeringa llena de mediano a la aguja nr.2 y compruebe si hay alguna fuga. Asegúrese de que las salidas de medianas de nr.1 aguja y la última sangre restante se perfunde fuera del corazón. </li></ol> 6. Coloque cámara de perfusión en el Stand <ol><li> Llenar cámara de perfusión con los 20 ml de medio transferidos. Coloque la junta y la tapa de la cámara y apretar con lavadora y tornillos. </li><li> Retire los MTCS reunidos desde el INCUBAtor. Coloque la cámara de perfusión en el estrado. Conecte la tubería (ver Figura 1D). Coloque el soporte en la incubadora (5% de CO 2 y 37 ° C). Conectar el tubo a la bomba. Encienda la bomba con velocidad de flujo alrededor de 600 l / min. </li><li> Asegúrese de que en el depósito y / o la trampa de burbujas el nivel del medio permite que las visualizaciones de la presencia del flujo por la caída de las gotas medianas entrantes. Después del cultivo, el corazón es retirado del sistema, fijaron y procesaron para examen histológico. </li></ol>

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The authors declare no conflicts of interest.

Los pasos críticos en el cultivo de las válvulas de ratón cardíacas incluyen hacer el tiempo entre la extirpación del corazón desde el ratón y la ligadura en la cámara de perfusión tan corto como sea posible para asegurar la viabilidad y la ligadura de las agujas perpendicular a las válvulas para garantizar la dirección apropiada de la corriente de . Adicionalmente, control del caudal después de la ligadura en la cámara de perfusión sin medio asegura la inserción y la ligación de agujas apropiada. Es fun...

Enfermedad de las válvulas cardíacas son una causa importante de morbilidad y mortalidad en el mundo occidental; su prevalencia aumenta con la edad y afecta a más del 10% de la población de 75 años y mayores 1. Las válvulas de la parte sistémica del corazón, las válvulas aórtica y mitral, son en su mayoría afectada. Enfermedad de la válvula cardíaca se caracteriza por la pérdida de la estructura altamente organizada de las válvulas, lo que resulta en la alteración de las propiedades mecánicas 2. La integridad estructural tanto, es fundamental para la función de la válvula. Las valvas de la válvula se componen de células intersticiales valvulares (VIC), células endoteliales valvulares (VEC), y la matriz extracelular, que está altamente organizado en un patrón en capas 3,4. Los VIC son responsables de la síntesis de ECM, la degradación y la organización. Factores que emana de la circulación sanguínea, EC o resida en el acto de ECM en los VIC orquestar su función. En adición,fuerzas mecánicas actúan sobre la valva durante el ciclo cardíaco que resulta en laminar o tensión de cizallamiento oscilatorio, tensiones de compresión que influyen en la resistencia a la tracción o comportamiento de VIC 5. Para entender cómo se regula la estructura de la válvula, primero hay que entender cómo VIC responden a la diversa serie de estímulos experimentados durante el ciclo cardíaco. Los estudios in vitro han sido muy informativo sobre las características y capacidades de las células valvulares. La respuesta de estas células in vitro, sin embargo, no siempre puede imitar con precisión la respuesta in vivo 6; por ejemplo, la respuesta de VIC a los estímulos depende de la presencia de ECs y la composición ECM 5. Además, la respuesta de las células a los estímulos valvulares depende de su ubicación específica en el prospecto 7. Además de los estímulos bioquímicos, el comportamiento de las células valvulares se determina por las fuerzas mecánicas que actúan on la válvula 8. Cada región de la válvula se somete a su propio conjunto específico de tensiones hemodinámicas. Aunque los modelos actuales ex vivo han demostrado que las fuerzas mecánicas son determinantes importantes de la estructura valvular 5, los mecanismos asociados aún no están claros. Mientras que los modelos in vivo han proporcionado información sobre los mecanismos moleculares que subyacen el desarrollo valvular 9,10, conocimientos sobre la biología valvular adultos sigue siendo difícil de alcanzar. Por lo tanto, un modelo in vivo ex flujo fue desarrollado en el que las válvulas cardíacas pueden cultivarse en su posición natural en el corazón durante un período prolongado de tiempo 11. Esto tiene la ventaja de que las válvulas se mantienen en su configuración natural y los VIC experimentan el mismo entorno que in vivo, por lo que las respuestas a los estímulos vics tan natural como sea posible. Además, la cultura de las válvulas en su posición natural en el corazón facilita sometiendo cada unoregión valvular a las tensiones hemodinámicas relevantes. En este modelo ex vivo, es decir, el sistema de tejido miniatura Cultura (MTCS), las válvulas se puede someter a diferentes estímulos bioquímicos y hemodinámicos que permiten la investigación de su papel en la remodelación de la válvula cardiaca.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400-045</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-06</td><td></td></tr><tr><td>Silk 7-0</td><td>Ethicon</td><td>768G</td><td></td></tr><tr><td>100 mm culture dish</td><td>Greiner bio-one</td><td>664160</td><td></td></tr><tr><td>50 ml tube</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>5 ml syringe</td><td>BD</td><td>309649</td><td></td></tr><tr><td>21 G needle</td><td>BD</td><td>304432</td><td></td></tr><tr><td>Heparin</td><td>LEO</td><td>012866-08</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Micro Scissors, Economy, Vannas-type</td><td>Tedpella</td><td>1346</td><td></td></tr><tr><td>Silicon tubing</td><td>Thermo Scientific</td><td>8060-0020</td><td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td></tr><tr><td>Silicon tubing for pump</td><td>Masterflex</td><td>96400-13</td><td>I.D. x O.D. x Wall: 0.8 x1.59 x 0.40 mm</td></tr><tr><td>Biocompatible glue (Histoacryl)</td><td>B. Braun</td><td>1050071</td><td></td></tr><tr><td>precision vaporizer</td><td>Dr&amp;auml;ger</td><td>Vapor 200</td><td></td></tr><tr><td>peristaltic roller pump</td><td>Masterflex</td><td>7521-35</td><td></td></tr><tr><td>Easy-load pump head</td><td>Masterflex</td><td>7518-00</td><td></td></tr><tr><td>Flow chamber</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr><tr><td>Bubble trap</td><td></td><td></td><td>see Lieber &lt;i&gt;et al.,&lt;/i&gt; 2010</td></tr></tbody></table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

La válvula aórtica (Figura 2) o la válvula mitral 11 pueden ser cultivadas durante al menos 3 días. Mediante el cultivo en la posición abierta (que representa la posición sistólica de la válvula aórtica y la posición diastólica para la válvula mitral), las células permanecen viables valvulares. No se observa la muerte celular tal como se determina por la ausencia de células TUNEL positivas (Figura 2H, I) o exfoliados caspasa-3 de expresión (que no se muestra y ...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

El cultivo de ratón cardíaca válvulas en el sistema de cultivo tisular miniatura

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.8K vistas.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Video: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

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.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

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.

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

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

Investigación

Ingeniería

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

El trasplante de la válvula pulmonar Utilizando un modelo de ratón de heterotópico trasplante cardiaco

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Medicina

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 ...

Biología

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

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

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

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

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

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

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Modelo In Vitro de Angiogénesis Coronaria

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Aislamiento de las células murinas de la válvula endoteliales

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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

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

Aislamiento, cultivo y la transducción de cardiomiocitos de ratones adultos

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

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Aislamiento, Transfección y Cultura a Largo Plazo de Cardiomiocitos adultos de ratón y rata

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Aislamiento De Células De La Válvula Intersticial De Ratón Para Estudiar La Calcificación De La Válvula Aórtica In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Source: Bouchareb, R. et al., <a href="https://app.jove.com/v/62419">Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.</a>&#160; J. Vis. Exp. &#160;(2021).
This video demonstrates an assay technique for assessing the calcification of the aortic valve interstitial cells in vitro. The assay helps to quantify the calcium concentration using the arsenazo III reagent, a calcium-sensitive dye that reacts with calcium ions to form a purple-colored calcium-arsenazo complex.

Ensayo de calcificación para medir la concentración de calcio en las células intersticiales de la válvula aórtica

Source: Bouchareb, R. et al., Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.&#160; J. Vis. Exp. ...

El cultivo de ratón cardíaca válvulas en el sistema de cultivo tisular miniatura

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