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

Questo protocollo segue le linee guida LUMC dell&#39;animale comitato etico della ricerca. 1. Preparazione degli strumenti, Cultura Media e MTC Nota: eseguire tutti i preparativi in ​​cappa a flusso laminare. La MTC camera di perfusione, trappola per bolle e supporto sono descritti in Lieber et al., 2010 11. <ol><li> Disinfettare le pinze e forbici micro con il 70% di etanolo. Preparare una siringa da 5 ml con 21G ago con tampone (130 NaCl sterile Tyrode mM, 5,4 mM KCl, 6.0 mM Hepes, 1.2 MgSO mm 4, 1,2 mm KH 2 PO 4, 20 Glucosio mM, 1,5 mM CaCl 2 .2H 2 O, pH = 7.2). Preparare una siringa da 5 ml con ago 21G con la soluzione KCl sterile (100 mm). </li><li> Preparare 65 ml di terreno per cuore: DMEM supplementato con 10% siero fetale bovino, antibiotici / antimicotici (A / A; 100 unità di penicillina, 100 mg di streptomicina, e 0,25 ug di amfotericina B / ml), insulino-Transferrin-Selenio (ITS; 10 mg / ml di insulina, 5.5 mg / ml transferrina, 6.7 ng / ml selenito di sodio) e filtro sterilizzare. </li><li> Sterilizzare la camera di perfusione e gorgogliatore spruzzando con il 70% di etanolo e asciugare con carta assorbente. Assemblare la MTC (Figura 1D), tuttavia, inizialmente senza la camera di perfusione. Posizionare la trappola bolla sul cavalletto. Utilizzare una pompa peristaltica a rulli con testate pompa facile carico. <ol><li> Utilizzando tubo in silicone effettuare le connessioni tra il serbatoio (tubo da 50 ml) e la pompa, la pompa e il gorgogliatore, il gorgogliatore e serbatoio (Figura 1D). Rendere il tubo all&#39;interno dell&#39;incubatrice abbastanza lungo da permettere medio raggiungimento dell&#39;equilibrio gas con il gas nell&#39;incubatrice attraverso il tubo di gas permeabile silicio (1 m) e il tubo all&#39;esterno dell&#39;incubatrice più breve possibile. </li></ol></li><li> Riempire il tubo da 50 ml con il 70% di etanolo, accendere la pompa (velocità del flusso intorno a 1 ml / min per consentire la corretta circolazione) e lasciare che il CIR etanolocolare per 30 min per sterilizzare tutti i tubi. Rimuovere l&#39;etanolo svuotando il serbatoio e pompando l&#39;etanolo dal sistema. </li><li> Riempire il tubo da 50 ml di acqua distillata sterile, accendere la pompa e lasciare circolare l&#39;acqua per 30 minuti per sbarazzarsi di etanolo residuo. Rimuovere l&#39;acqua svuotando il serbatoio e pompare l&#39;acqua dal sistema. </li><li> Riempire il tubo da 50 ml con 45 ml di terreno, mettere lo stand nell&#39;incubatrice. Accendere il (velocità di flusso di circa 1 ml / min) e pompa circolare il mezzo di riempire tutti i tubi, rimuovere tutte le bolle d&#39;aria e permettere il mezzo di adattarsi alla composizione del gas nell&#39;incubatrice per almeno 1 ora. Mantenere l&#39;incubatore a condizioni di coltura tissutale-standard (5% di CO 2 e 37 ° C). Assicurarsi che non siano presenti bolle nel tubo. </li></ol> 2. Isolamento di Cuore mouse <ol><li> Iniettare il mouse con 500 unità di eparina. Ciò impedirà la coagulazione del sangue e consente la rimozione di blood dal cuore. Dopo 10 min, anestetizzare il mouse in una camera di induzione con 4% isoflurano utilizzando un vaporizzatore di precisione. L&#39;anestesia è sufficiente quando il mouse non risponde a pizzico piedi. </li><li> Trasferire il mouse per una tavola di dissezione e mantenere l&#39;anestesia mediante una maschera facciale collegato ad un circuito coassiale. Disinfettare la pelliccia del mouse con il 70% di alcol. Utilizzando le forbici aprire la cavità addominale per esporre la vena cava e il diaframma. </li><li> Per esporre il cuore, fare incisioni laterali a partire dall&#39;ultimo al primo costole e riflettono la gabbia toracica sopra la testa di topo. Arrestare il anestesia come mouse non respira più. Osservare il battito del cuore. </li><li> Inserire l&#39;ago 21G attaccato ad una siringa da 5 ml contenente tampone sterile Tyrode nella vena cava inferiore dalla addominale nella cavità toracica (attraverso diaframma). Assicurarsi che il sangue possa uscire dalla vena cava caudale dall&#39;inserzione dell&#39;ago. Profumato °Buffer e Tyrode delicatamente e con pressione costante nella vena cava fino a quando il cuore perde in parte il suo colore rosso. Nota: Il cuore rimane battere permettendo la rimozione del sangue dal cuore. </li><li> Sostituire l&#39;ago con l&#39;ago 21G attaccato ad una siringa da 5 ml contenente soluzione KCl sterile. Profumato delicatamente la soluzione KCl nella vena cava fino a quando il cuore smette di battere e rimuovere l&#39;ago. Nota: La soluzione KCl preserva cuore nella fase diastolica rilassata, che consentirà più facile inserimento degli aghi e perfusione perfusione del sistema coronarico. </li><li> Sollevare leggermente il cuore con pinze curve e con le forbici sezionano il cuore libero dal tessuto circostante, ma lasciano arterie e le vene prossimali al cuore intatto e collegato al cuore. Trasferire il cuore ad una provetta da 15 ml contenente PBS ghiacciato integrato con antibiotici / antimicotici (A / A). Conservare il cuore in PBS freddo per un massimo di 3 ore. </li></ol> 3. Cannulation dei Cuori mouse nella camera di perfusione <ol><li> Eseguire tutte le procedure di cappa a flusso laminare. Trasferire il cuore isolato dal tubo 15 ml per un piatto di 10 centimetri di Petri e aggiungere PBS integrato con A / A. </li><li> Sotto un microscopio da dissezione, utilizzare il micro forbice e pinze per rimuovere tutto il tessuto non cardiaca ma preservare l&#39;aorta ascendente almeno alla biforcazione con l&#39;arteria anonima e le vene polmonari, 2 mm prossimale al cuore. Tagliare la punta della punta del cuore per creare l&#39;accesso al lume del ventricolo sinistro (tipicamente 2 mm). Posizionare la camera di perfusione nella cappa a flusso sotto il microscopio da dissezione. </li><li> Attaccare una siringa da 5 ml con il mezzo all&#39;ago inserto utilizzando il tubo in silicone. Riempire la camera di perfusione con 20 ml di mezzo e mettere il cuore sul palco di rotazione tra i 2 aghi smussati nella camera di perfusione (Figura 1). Regolare l&#39;altezza della fase di rotazione in modo che il cuore è posizionato di fronte allaaghi. </li></ol> 4. legatura per la coltura della valvola mitrale (vedi Figura 1) <ol><li> Legare l&#39;aorta con sutura (seta 7.0). Legare l&#39;appendice atriale sinistra con sutura. Inserire l&#39;ago (nr. 1 in figura 1) attraverso la vena polmonare in atrio sinistro e legare con sutura prossimale alla sua entrata nell&#39;atrio sinistro. Assicurarsi che l&#39;ago non è troppo lontano in atrio sinistro in quanto ciò danneggerebbe la valvola mitrale. </li><li> Inserire l&#39;ago (nr.2 in figura 1) con moto lineare nel ventricolo sinistro. Trasferire il terreno dalla camera di perfusione in una provetta da 50 ml. Sigillare l&#39;ago al miocardio con colla biocompatibile. </li><li> Dopo che la colla si è asciugata, iniettare attentamente medio nel cuore collegando una siringa riempita di medio all&#39;ago nr.1 e verificare se vi siano perdite. Garantire che le uscite medie di ago nr.2 e l&#39;ultimo sangue rimanente viene perfuso dal cuore. </li></ol> 5. Ligazione per la coltura della valvola aortica (vedi Figura 1) <ol><li> Inserire l&#39;ago (nr.1 in figura 1) nell&#39;aorta e legare con suture. Assicurarsi che l&#39;ago non è troppo lontano in aorta poiché ciò danneggiare la valvola aortica. Inserire l&#39;ago (nr.2 in figura 1) con moto lineare nel ventricolo sinistro. Trasferire il terreno dalla camera di perfusione in una provetta da 50 ml. Sigillare l&#39;ago al miocardio con colla biocompatibile. </li><li> Dopo che la colla si è asciugata, iniettare attentamente medio nel cuore collegando una siringa riempita di medio all&#39;ago nr.2 e verificare se vi siano perdite. Garantire che le uscite medie di ago nr.1 e l&#39;ultimo sangue rimanente viene perfuso dal cuore. </li></ol> 6. Mettere Perfusion Camera Stand <ol><li> Riempire camera di perfusione con i 20 ml trasferiti medio. Mettere guarnizione e coperchio sulla camera e stringere con rondella e viti. </li><li> Rimuovere i MTC assemblati dal incubazionetor. Fissare la camera di perfusione sul cavalletto. Collegare il tubo (vedi Figura 1D). Posizionare il supporto in incubatrice (5% di CO 2 e 37 ° C). Collegare il tubo alla pompa. Accendere la pompa con velocità del flusso circa 600 ml / min. </li><li> Assicurarsi che nel serbatoio e / o il gorgogliatore il livello del fluido consente le visualizzazioni della presenza del flusso dalla caduta delle gocce medie in entrata. Dopo coltura, il cuore viene rimosso dal sistema, fissi ed elaborati per l&#39;esame istologico. </li></ol>

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	<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.

Fasi critiche coltivando le valvole cardiache di topo comprendono rendendo il tempo tra escissione del cuore dal mouse e la legatura nella camera di perfusione più breve possibile per la sussistenza e legatura degli aghi perpendicolare alle valvole per assicurare la corretta direzione del flusso . Inoltre, il controllo della portata dopo legatura nella camera di perfusione senza prodotto garantisce il corretto inserimento e legatura di aghi. È fondamentale mantenere una cultura sterile, di evitare bolle d&#39;aria nel...

Malattia della valvola cardiaca è una delle principali cause di morbilità e mortalità nel mondo occidentale; la sua prevalenza aumenta con l&#39;età e colpisce oltre il 10% della popolazione di 75 anni e più 1. Le valvole della parte sistemica del cuore, le valvole aortica e mitrale, sono per lo più colpite. Malattia della valvola cardiaca è caratterizzata dalla perdita della struttura altamente organizzata delle valvole, che provoca l&#39;alterazione delle proprietà meccaniche 2. L&#39;integrità strutturale è quindi cruciale per il funzionamento della valvola. Le foglioline della valvola sono costituiti da cellule interstiziali valvolari (VIC), cellule endoteliali valvolari (VEC) e matrice extracellulare, che è altamente organizzato in un modello a strati 3,4. I VIC sono responsabili della sintesi ECM, degradazione e organizzazione. Fattori che emana dal flusso sanguigno, EC o residenti in atto ECM sulle VIC orchestrare la sua funzione. In aggiunta,forze meccaniche del foglio durante il ciclo cardiaco conseguente laminare o sollecitazione di taglio oscillatoria, sollecitazioni di compressione o di trazione che influenzano il comportamento di VIC 5. Per comprendere come è regolata la struttura della valvola, deve prima essere compreso come VIC rispondere alle diverse serie di stimoli sperimentati durante il ciclo cardiaco. Studi in vitro sono stati molto informato circa le caratteristiche e le abilità delle cellule valvolari. La risposta di queste cellule in vitro, tuttavia, non sempre simulare accuratamente la risposta in vivo 6; per esempio, la risposta agli stimoli VIC dipende dalla presenza di cellule endoteliali e la composizione ECM 5. Inoltre, la risposta delle cellule valvolari agli stimoli dipende dalla loro posizione specifica nel foglietto 7. Oltre agli stimoli biochimici, il comportamento delle cellule valvolari è determinata da forze meccaniche o agisconon la valvola 8. Ogni regione della valvola è sottoposto a un insieme specifico di sollecitazioni emodinamiche. Sebbene attuali ex vivo i modelli hanno dimostrato che le forze meccaniche sono importanti determinanti della struttura valvolare 5, i meccanismi associati sono ancora poco chiari. Mentre i modelli in vivo hanno fornito informazioni sui meccanismi molecolari alla base dello sviluppo valvolare 9,10, intuizioni adulto valvolare biologia è ancora sfuggente. Pertanto, un modello in vivo ex flusso è stato sviluppato in cui le valvole cardiache possono essere coltivati ​​nella loro posizione naturale nel cuore per un periodo di tempo prolungato 11. Questo ha il vantaggio che le valvole rimangono nella loro configurazione naturale e le VIC sperimentano lo stesso ambiente in vivo, rendendo le risposte VICS a stimoli più naturale possibile. Inoltre, la cultura delle valvole nella loro posizione naturale nel cuore facilita sottoponendo ogniregione valvolare alle relative sollecitazioni emodinamiche. In questo modello ex vivo, cioè, il Sistema Miniature Tissue Culture (MTC), le valvole possono essere sottoposto a diversi stimoli biochimici ed emodinamici permettendo l&#39;indagine del loro ruolo nel rimodellamento della valvola 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 valvola aortica (Figura 2) o valvola mitrale 11 possono essere coltivate per almeno 3 giorni. Coltivando in posizione aperta (che rappresenta la posizione sistolico della valvola aortica e la posizione diastolica per la valvola mitrale), cellule valvolari rimangono vitali. Nessuna morte cellulare è osservato come determinato dall&#39;assenza di TUNEL-positive cellule (Figura 2H, I) o spaccati caspasi-3 espressione (non mostrato e Lieber et al., 2010 11...

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.

Coltura mouse cardiaca Valvole in miniatura Tissue Culture Sistema

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 ...

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Bioengineering

8.8K Views.  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.

Coltura in vitro di cellule provenienti da epicardici Cuore embrionali di topo

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

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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.

Il trapianto di valvola polmonare Utilizzando un modello murino di trapianto cardiaco eterotopico

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; Metodo di Cultura per il mouse embrionale del cuore

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 del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

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.

Modello in vitro dell'angiogenesi coronarica

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.

Isolamento di cellule murine endoteliali Valve

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 trasduzione di topo adulto cardiomiociti

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, trasfezione e cultura a lungo termine di cardiomiociti per topi e ratti adulti

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 delle celle della valvola interstiziale del topo per studiare la calcificazione della valvola aortica 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.

Test di calcificazione per misurare la concentrazione di calcio nelle cellule interstiziali della valvola aortica

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. ...

Coltura mouse cardiaca Valvole in miniatura Tissue Culture Sistema

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