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 segue as diretrizes LUMC do animal comitê de ética em pesquisa. 1. Preparação de Instrumentos, Meio de Cultura, e MTCS Nota: Execute todos os preparativos na capela de fluxo laminar. O MTCS câmara de perfusão, borbulhador e suporte são descritos na Lieber et al., 2010 11. <ol><li> Desinfectar os fórceps e micro tesoura com etanol 70%. Prepara-se uma seringa de 5 mL com uma agulha 21G com NaCl estéril de Tyrode Buffer (130 mM, KCl 5,4 mM, HEPES 6,0 mM, 1,2 MgSO 4 mM, 1,2 mM de KH 2 PO 4, glicose 20 mM, 1,5 mM de CaCl 2 .2H 2 O, pH = 7,2). Prepara-se uma seringa de 5 mL com uma agulha de 21G estéril com solução de KCl (100 mM). </li><li> Preparar 65 ml de meio por coração: DMEM suplementados com 10% de Soro Fetal de Vitela, antibiótico / antimicótico (A / A; 100 unidades de penicilina, 100 ug de estreptomicina, e 0,25 ug de anfotericina B / ml), insulina-Transferrin-O selénio (STI; 10 ug / ml de insulina, 5,5 ng / ml de transferrina, 6,7 ng / ml de selenito de sódio) e esterilizar filtro. </li><li> Esterilizar a câmara de perfusão e de retenção de bolhas por pulverização com 70% de etanol e seco com uma toalha de papel. Montar o MTCS (Figura 1D), no entanto, inicialmente sem a câmara de perfusão. Posicione a armadilha de bolhas no stand. Use uma bomba de roletes com cabeças de bomba peristáltica fácil de carga. <ol><li> Usando tubo flexível de silicone fazer ligações entre o reservatório (tubo de 50 ml) e a bomba, a bomba e a armadilha de bolhas, o borbulhador e reservatório (ver Figura 1D). Adicione a tubagem no interior da incubadora o tempo suficiente para permitir que forma a atingir o equilíbrio de gás com o gás na incubadora através da tubagem de gás permeável de silício (1 M) e o tubo exterior da incubadora tão curto quanto possível. </li></ol></li><li> Encha o tubo de 50 ml com etanol 70%, ligar a bomba (velocidade de fluxo de cerca de 1 ml / min para permitir a circulação adequada) e deixar o cir etanolcular por 30 min para esterilizar toda a tubulação. Remover o etanol por esvaziamento do reservatório e bombeando o etanol a partir do sistema. </li><li> Encha o tubo de 50 ml com água destilada estéril, ligar a bomba e deixe a água circule durante 30 minutos para se livrar do restante etanol. Remover a água por esvaziamento do reservatório e a bombear a água para fora do sistema. </li><li> Encha o tubo de 50 ml com 45 ml de meio, coloque o suporte na incubadora. Ligue o (velocidade de fluxo de cerca de 1 ml / min) e a bomba faz circular o meio para preencher toda a tubagem, remover todas as bolhas de ar e permitir que a forma para se adaptar à composição do gás na incubadora durante pelo menos 1 h. Manter a incubadora a condições-padrão de cultura de tecidos (5% CO 2 e 37 ° C). Assegure-se que não há bolhas de ar na tubagem. </li></ol> 2. Isolamento de rato coração <ol><li> Injectar o mouse com 500 unidades de heparina. Isto irá evitar a coagulação do sangue e permite a remoção de bLood a partir do coração. Após 10 min, anestesiar o rato numa câmara de indução com 4% de isoflurano utilizando um vaporizador de precisão. A anestesia é suficiente quando o mouse não responde aos pés pitada. </li><li> Transferir o rato para uma placa de dissecção e manter a anestesia por meio de uma máscara ligada a um circuito coaxial. Desinfectar a pele do rato com 70% de álcool. Com uma tesoura de abrir a cavidade abdominal para expor a veia cava e diafragma. </li><li> Para expor o coração, fazer incisões laterais a partir da última para a primeira costelas e refletem a caixa torácica sobre a cabeça de rato. Pare a anestesia como mouse não está respirando mais. Observe o coração batendo. </li><li> Inserir a agulha 21G ligada a uma seringa de 5 ml estéril contendo tampão de Tyrode para a veia cava inferior do abdominal para dentro da cavidade torácica (através do diafragma). Certifique-se de que o sangue pode sair da veia cava caudal a partir da inserção da agulha. Perfundir thTampão de Tyrode e suavemente e com uma pressão constante para a veia cava até que o coração perde parcialmente a sua cor vermelha. Nota: o coração permanece a bater, permitindo a remoção do sangue a partir do coração. </li><li> Substituir a agulha com a agulha 21G ligada a uma seringa de 5 ml contendo solução de KCl estéril. Suavemente perfundir a solução de KCl para a veia cava até o coração parar de bater e remover a agulha. Nota: A solução de KCl preserva o coração na fase diastólica relaxada, o que vai permitir uma inserção mais fácil das agulhas de perfusão e do sistema de perfusão coronária. </li><li> Levante ligeiramente o coração usando uma pinça curva e usando uma tesoura dissecar o coração livre do tecido circundante, mas deixar artérias e veias proximais ao coração intactos e anexado ao coração. Transferir o coração para um tubo de 15 ml contendo PBS arrefecido em gelo suplementado com antibiótico / antimicótico (A / D). Armazenar o coração em PBS gelado por até 3 horas. </li></ol> 3. Cannulation dos corações mouse na câmara de perfusão <ol><li> Efectuar todos os procedimentos em capela de fluxo laminar. Transferir o coração isolado a partir do tubo de 15 ml a um prato de 10 cm de Petri e adicionar PBS suplementado com A / A. </li><li> Sob um microscópio de dissecação, o uso de tesoura e micro fórceps para remover todo o tecido cardíaco, mas não preservar a aorta ascendente, pelo menos, da bifurcação com a artéria inominada e as veias pulmonares, 2 mm proximal ao coração. Cortar a extremidade da ponta do coração para criar um acesso para o lúmen do ventrículo esquerdo (tipicamente 2 mm). Coloque a câmara de perfusão em câmara de fluxo a sob o microscópio de dissecação. </li><li> Anexar uma seringa de 5 ml com meio à agulha de inserção usando o tubo de silicone. Encher a câmara de perfusão com 20 ml de meio e colocar o coração no estágio de rotação entre as agulhas embotadas 2 na câmara de perfusão (Figura 1). Ajustar a altura da fase de rotação de modo que o coração está posicionada em frente doagulhas. </li></ol> 4. A ligadura para a cultura das valva mitral (ver figura 1) <ol><li> Ligadura da aorta com sutura (seda 7.0). Ligadura do apêndice atrial esquerdo com sutura. Insira a agulha (nr. 1 na Figura 1) através da veia pulmonar para o átrio esquerdo e ligar com sutura proximal à sua entrada no átrio esquerdo. Certifique-se de que a agulha não é muito longe para o átrio esquerdo, pois isso iria danificar a válvula mitral. </li><li> Inserir a agulha (nr.2 na Figura 1) com um movimento linear para o ventrículo esquerdo. Transferir o meio a partir da câmara de perfusão para um tubo de 50 ml. Selar a agulha para o miocárdio com cola biocompatível. </li><li> Depois que a cola secar, injetar cuidadosamente em meio coração, ligando uma seringa cheia de médio a agulha nr.1 e verifique se há algum vazamento. Assegurar que as saídas médias de nr.2 agulha eo último sangue restante é perfundido para fora do coração. </li></ol> 5. Ligação para a cultura das Válvula Aórtica (veja a Figura 1) <ol><li> Inserir a agulha (nr.1 na Figura 1) na aorta e ligar com sutura. Certifique-se de que a agulha não é muito longe para a aorta como isso iria danificar a válvula aórtica. Inserir a agulha (nr.2 na Figura 1) com um movimento linear para o ventrículo esquerdo. Transferir o meio a partir da câmara de perfusão para um tubo de 50 ml. Selar a agulha para o miocárdio com cola biocompatível. </li><li> Depois que a cola secar, injetar cuidadosamente médio para o coração, ligando uma seringa cheia de médio a agulha nr.2 e verifique se há algum vazamento. Assegurar que as saídas médias de nr.1 agulha eo último sangue restante é perfundido para fora do coração. </li></ol> 6. Coloque Perfusão Secção de suporte <ol><li> Encha câmara de perfusão com os 20 ml de meio transferidos. Coloque a junta e tampa na câmara e aperte com lavadora e parafusos. </li><li> Remova os MTCS montados a partir do incubator. Fixe a câmara de perfusão no stand. Conecte o tubo (veja a Figura 1D). Colocar o suporte na incubadora (5% de CO 2 e 37 ° C). Conecte a tubulação à bomba. Ligue a bomba com velocidade de fluxo de cerca de 600 mL / min. </li><li> Certifique-se de que no reservatório e / ou o borbulhador o nível do meio permite que as visualizações da presença do fluxo pela queda das gotas médias de entrada. Após a cultura, o coração é removido do sistema, fixadas e processadas para exame histológico. </li></ol>

<ol>
	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

As etapas críticas em cultura das válvulas cardíacas de rato incluem fazer o tempo entre a excisão do coração do rato e a ligação na câmara de perfusão tão curto quanto possível, para assegurar a viabilidade e ligação das agulhas perpendicular às válvulas para assegurar a direcção correcta de fluxo . Além disso, a verificação do fluxo após a ligadura na câmara de perfusão sem forma garante uma inserção correcta e ligadura de agulhas. É essencial para manter uma cultura estéril e evitar bolhas...

Doença valvar cardíaca é uma das principais causas de morbidade e mortalidade no mundo ocidental; sua prevalência aumenta com a idade e afeta mais de 10% da população de 75 anos ou mais 1. As válvulas da parte sistêmica do coração, as valvas aórtica e mitral, são os mais afetados. Válvula cardíaca doença é caracterizada pela perda da estrutura altamente organizada das válvulas, o que resulta na alteração das propriedades mecânicas 2. A integridade estrutural é, portanto, crucial para a função da válvula. Os folhetos da válvula são constituídos por células intersticiais valvulares (VIC), células endoteliais valvulares (VEC), e matriz extracelular, o que é altamente organizado num padrão em camadas 3,4. Os VICs são responsáveis ​​pela síntese de ECM, degradação e organização. Fatores que emana da corrente sanguínea, ECs ou residentes no ato ECM nas VICs orquestrando sua função. Além,forças mecânicas agir sobre o folheto durante o ciclo cardíaco, resultando em laminar ou tensão de cisalhamento oscilatório, tensões de compressão ou de tracção que influenciam o comportamento de VICs 5. A fim de compreender como a estrutura da válvula é regulado, ele deve primeiro ser compreendida como VICs responder ao conjunto diversificado de estímulos experimentadas durante o ciclo cardíaco. Estudos in vitro têm sido muito informativo sobre as características e capacidades das células valvulares. A resposta destas células, in vitro, no entanto, nem sempre podem mimetizar com exactidão a resposta in vivo 6; Por exemplo, a resposta a estímulos de VIC é dependente da presença de células endoteliais e a composição 5 ECM. Além disso, a resposta das células a estímulos valvulares depende da sua localização específica no folheto 7. Além disso a estímulos bioquímicos, o comportamento das células valvulares é determinada por forças mecânicas que actuam ón a válvula 8. Cada região da válvula é submetida a seu próprio conjunto específico de tensões hemodinâmicas. Embora atuais modelos ex vivo têm mostrado que as forças mecânicas são importantes determinantes da estrutura valvular 5, os mecanismos associados ainda não estão claros. Enquanto os modelos in vivo têm fornecido insights sobre os mecanismos moleculares subjacentes ao desenvolvimento valvular 9,10, insights sobre biologia valvular adulto ainda é indescritível. Por conseguinte, um modelo ex vivo de fluxo foi desenvolvido em que as válvulas cardíacas podem ser cultivadas na sua posição natural no coração por um período prolongado de tempo 11. Isto tem a vantagem de que as válvulas permanecem na sua configuração natural e os VICs experimentar o mesmo ambiente como in vivo, fazendo com que as respostas a estímulos VICs tão natural quanto possível. Além disso, a cultura das válvulas na sua posição natural no coração facilita a sujeição de cada umregião valvular às tensões hemodinâmicas relevantes. Neste modelo ex vivo, isto é, o sistema de cultura de tecidos em miniatura (MTCS), as válvulas podem ser submetidos a diferentes estímulos bioquímicos e hemodinâmicas, permitindo o estudo do seu papel na remodelação da válvula cardíaca.

<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.

A válvula da aorta (Figura 2) da válvula mitral ou 11 podem ser cultivadas por pelo menos 3 dias. Através da cultura na posição aberta (que representa a posição sistólica para a válvula aórtica diastólica e a posição da válvula mitral para a), as células permanecem viáveis ​​valvulares. Não é observada a morte celular como determinado pela ausência de células de TUNEL positivo (Figura 2H, I) ou clivado expressão da caspase-3 (não mostrado e Lieber <e...

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.

Cultura Rato Cardiac Valves no Sistema de Cultura de Tecidos Miniature

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 visualizações.  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.

Vídeo: 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.

Cultura in vitro de células epicárdicas De Coração embrionárias de camundongos

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

Engenharia

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.

O transplante de válvula pulmonar Usando um modelo do rato de Heterotópica Transplante de Coração

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 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 ...

Biologia

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 ...

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 Angiogênese Coronariana

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.

O isolamento de células endoteliais de Murino Válvula

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.

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 ...

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.

Isolamento, Transfecção e Cultura de Longo Prazo de Cardiomiócitos de Ratos Adultos e Ratos

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.

Isolamento de células válvulas intersticiais do rato para estudar a calcificação da 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.

Ensaio de calcificação para medir a concentração de cálcio em células intersticiais da 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. ...

Cultura Rato Cardiac Valves no Sistema de Cultura de Tecidos Miniature

Resumo

Explore mais vídeos

Capítulos neste vídeo