Cette recherche est soutenue par la bourse de carrière NSF, la Fondation Hartwell, et l&#39;American Heart Association (# 0830384N).

1. Préparation <ol><li> Autoclave dans un instrument couvert plateau les éléments suivants: <ol><li> Pince tissus dentelé - Pour la manipulation des tissus dépliant </li><li> Ciseaux Tissu (8 cm) - Pour couper le tissu dépliant et les cuspides </li><li> Cotons-tiges - Pour isoler la couche endothéliale de la notice ou aube </li></ol></li><li> Faire solution de collagénase stérile <ol><li> Ajouter 4,0 g de poudre DMEM à 250 ml de 18 MQ eau. </li><li> Ajouter 1,11 g de bicarbonate de sodium. </li><li> Ajouter (600 U / ml) 180 000 unités de collagénase. </li><li> Ajouter 1% (3 ml) de pénicilline / streptomycine. </li><li> Ajuster le pH de la solution à 7,2. </li><li> Porter la solution à 270 ml. </li><li> Stériliser la solution en passant par un filtre de 0,2 um. </li><li> Ajouter 10% (30 ml) de solution stérile de sérum de veau foetal. </li></ol></li><li> Faire stériles moyennes des cellules endothéliales <ol><li> Ajouter 6,7 grammes de poudre de DMEM à 400 ml d&#39;eau 18 MQ. </li><li> Ajouter 1,85 g de bicarbonate de sodium. </li><li> Ajouter (50 U / ml) 25 000 unités d&#39;héparine. </li><li> Ajouter 1% (5 ml) de pénicilline / streptomycine. </li><li> Ajuster le pH de la solution à 7,2. </li><li> Porter la solution à 450 ml. </li><li> Stériliser la solution en passant par un filtre de 0,2 um. </li><li> Ajouter 10% (50 ml) de solution stérile de sérum de veau foetal. </li></ol></li><li> Faire stériles moyennes des cellules interstitielles <ol><li> Ajouter 13,37 grammes de poudre DMEM à 800 ml de 18 MQ eau. </li><li> Ajouter 3,7 grammes de bicarbonate de sodium. </li><li> Ajouter 1% (10 ml) de pénicilline / streptomycine. </li><li> Ajuster le pH de la solution à 7,2. </li><li> Porter la solution à 900 ml. </li><li> Stériliser la solution en passant par un filtre de 0,2 um. </li><li> Ajouter 10% (100 ml) de sérum bovin stérile. </li></ol></li></ol> 2. Isolement des feuillets de la valve cardiaque <ol><li> Accise la racine aortique immédiatement et de façon aseptique du cœur après le sacrifice. </li><li> Rincer soigneusement la racine aortique de sang avec le froid DPBS stérile. Il est impératif d&#39;enlever tous les composants sanguins dès que possible pour limiter la mort VEC et la contamination bactérienne. Les antibiotiques et les antifongiques ne sont pas informés à ce stade car ils sont potentiellement dangereux pour la CVE. </li><li> Isoler feuillets de la valve (3 par soupape) directement à partir de la racine et la placer dans un tube de 15 ml stériles coniques remplis avec 12 ml DPBS froid. Agiter plusieurs fois pour enlever les débris et remplir avec du DPBS frais. </li><li> Transport au laboratoire sur la glace. </li><li> Dès l&#39;arrivée au laboratoire, placer le récipient avec le tissu sous la hotte stérile. </li></ol> 3. L&#39;isolement de couche endothéliale <ol><li> Remplir un plat stériles 35mm avec 3 ml de solution de collagénase froid par la vanne (3 feuillets). </li><li> Placez tous les trois folioles du tube 15 ml dans le plat rempli de la solution de collagénase. </li><li> Incuber les tissus pendant 5-10 minutes à 37 ° C. </li><li> Retirer délicatement la couche endothéliale par rotation d&#39;un écouvillon sec stérile sur la surface de la notice. Le sens de rotation et la quantité de cisaillement appliquée est essentielle pour la pureté de votre échantillon. La rotation de l&#39;écouvillon doit être dans une direction opposée au mouvement linéaire de votre main la création d&#39;un cisaillement contrôlé. Ce cisaillement est ce que soulève les cellules endothéliales du tissu. La quantité de force appliquée doit être suffisante pour sentir la résistance du tissu, mais ne pénètrent pas la membrane basale. </li><li> Occasionnellement, tamponnez l&#39;écouvillon dans la solution de la collagénase pour déloger les cellules à partir des fibres pointe. Après écouvillonnage est complète, la texture de la couche endothéliale doit se sentir légèrement plus lisse qu&#39;avant. </li><li> Recueillir la suspension cellulaire / collagénase et le transfert dans un nouveau tube de 15 ml stérile. </li><li> Centrifuger les tubes à 1000 tpm pendant 5 minutes pour culotter les cellules isolées et aspirer le surnageant. Si isoler des cellules interstitielles ainsi, effectuer ce protocole alors que ces cellules sont centrifugées. </li><li> Ajouter 3 mL de milieu de porc à l&#39;endothélium des tubes de 15 ml, centrifuger une deuxième fois, et les médias aspirer. Cette seconde centrifugation permet de filtrer certains des matériaux indésirables tels que les fibres pointe. </li><li> Re-suspendre les cellules endothéliales centrifugé dans 5 ml de milieu porcine endothéliales et les cellules plaque dans un pré-enduites T-25 flacon avec du collagène (utiliser une fiole jaugée par tube de centrifugation). </li><li> Laissez les cellules se développent au moins 2-3 jours avant de changer le support endothéliales. Cela permet de récupérer les cellules et se divisent depuis le processus d&#39;isolement est assez dur et le rendement cellulaire peut être faible. Il est essentiel de cellules passage près confluent depuis l&#39;inhibition de contact pourrait conduire à la transformation cellulaire. </li></ol> 4. Préparation de 60 mm pour boîte de culture isolement spécifique Side <ol><li> Ligne du verre de 60 mm des boîtes de Pétri avec du papier aluminium (2 plats en verre par leaflet). La feuille d&#39;aluminium permet d&#39;enlever la paraffine, de sorte que le plat en verre de Pétri peuvent être réutilisés pour d&#39;autres isolements. </li><li> Placez des perles de paraffine dans les plats, à moitié plein, et la couverture pour l&#39;autoclavage. </li><li> Une fois l&#39;autoclave est complète et la paraffine est fondue, déplacer l&#39;ensemble plat sur une surface plane fraîche. Comme la paraffine se refroidit, il durcit et créer une couche qui soutiendra piqûres d&#39;aiguille. </li><li> Après 30 minutes, le plat stérilisée peut alors être utilisé comme une chambre d&#39;isolement pour immobiliser le tissu notice. </li></ol> 5. Isolement du côté couche endothéliale spécifiques <ol><li> Retirez les tracts des tubes de 15 ml et placer sur le plat de culture préparés pour l&#39;isolement spécifiques 60mm côté. </li><li> Manipuler le dépliant pour que le côté ventricularis est face cachée sur la surface de la paraffine en laissant le côté fibrosa exposés. Epingler les bords de la notice pour exposer la couche endothéliale. </li><li> Verser quelques gouttes de la collagénase froid sur chaque surface (à la hausse face) endothéliales et incuber les tissus pendant 5-10 minutes à 37 ° C. </li><li> Comme avant, retirez délicatement la couche endothéliale par rotation d&#39;un écouvillon sec stérile sur la surface de la notice. Le sens de rotation et la quantité de cisaillement appliquée est essentielle pour la pureté de votre échantillon. La rotation de l&#39;écouvillon doit être dans une direction opposée au mouvement linéaire de votre main la création d&#39;un cisaillement contrôlé. Ce cisaillement est ce que soulève les cellules endothéliales du tissu et rien d&#39;autre. La quantité de force appliquée doit être suffisante pour sentir la résistance du tissu, mais ne pénètre pas dans le tissu. </li><li> Occasionnellement, tamponnez l&#39;écouvillon dans la solution de la collagénase pour déloger les cellules à partir des fibres pointe. Après écouvillonnage est complète, la texture de la couche endothéliale doit se sentir légèrement plus lisse qu&#39;avant. </li><li> Recueillir la suspension cellulaire / collagénase et le transfert dans un nouveau tube de 15 ml stérile. Indiquez une spécificité à côte sur l&#39;étiquette (tracts de la même valve peut être mis en commun). </li><li> Transfert des tracts à une boîte de culture nouvelle sorte que le côté ventricularis est maintenant exposée et répétez les étapes (05/03 au 05/06). </li><li> Une fois que tous suspension cellulaire / collagénase est recueillie, centrifuger les tubes à 1000 rpm pendant 5 minutes pour culotter les cellules isolées et aspirer le surnageant. Si isoler des cellules interstitielles ainsi, effectuer ce protocole alors que ces cellules sont centrifugées. </li><li> Ajouter 3 mL de milieu porcine endothéliales dans les tubes, centrifuger une deuxième fois, et aspirer les médias. Cette seconde centrifugation permet de filtrer certains des matériaux indésirables tels que les fibres pointe. </li><li> Re-suspendre les cellules endothéliales centrifugé dans 5 ml de milieu porcine endothéliales et les cellules plaque dans un pré-enduites T-25 fiole avec montage (utilisez une fiole jaugée par tube de centrifugation). </li><li> Laissez les cellules se développent au moins 2-3 jours avant de changer le support endothéliales. Cela permet de récupérer les cellules et se divisent, depuis le processus d&#39;isolement est assez sévère. Si vous remarquez le rendement est très faible, envisager de passer à un petit flacon ou d&#39;une plaque bien depuis adhérence cellule-cellule favorise la croissance cellulaire. N&#39;oubliez pas de passage près confluent depuis l&#39;inhibition de contact pourrait conduire à la transformation cellulaire. </li></ol> 6. Isolement des cellules interstitielles <ol><li> Remplissez un tube à centrifuger stérile de 15 ml avec 10 ml de solution de collagénase par soupape (3 feuillets). </li><li> Après écouvillonnage les tracts des cellules endothéliales, les placer dans le tube de 15 ml appropriée avec la solution de la collagénase. </li><li> Incuber pendant environ 12 à 18 heures (agiter délicatement si désiré). </li><li> Mélanger délicatement le tissu dégradé avec une pipette sérologique avant la suspension de cellules / collagénase devient homogène. Cette homogénéisation contribue à fragmenter le tissu et la libération des cellules interstitielles. </li><li> Centrifuger les tissus digérés pendant 5 minutes à 1000 rpm et aspirer le surnageant. </li><li> Ajouter 5 ml interstitielle moyenne porcine aux tubes de 15 ml, centrifuger une deuxième fois, et aspirer le liquide surnageant. </li><li> Re-suspendre les cellules endothéliales centrifugé dans 5 ml de milieu porcine interstitielle et la plaque de cellules dans un flacon T-75 (utiliser une fiole jaugée par tube de centrifugation). </li><li> Laissez les cellules se développent au moins 1-2 jours avant de changer le milieu cellulaire interstitiel. Il y aura des débris de tissu beaucoup plus que les cellules endothéliales, mais qui est attendu. Les cellules devraient également croître au confluent plus vite que les cellules endothéliales en raison de la cellule de rendement et de leur nature. </li></ol> 7. Images représentant <img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Figure 1" /> Figure 1. La morphologie des cellules isolées à 2-3 jours après l&#39;isolement. (A) VEC présentent une morphologie typique endothéliales et forment des grappes pour favoriser la croissance. (B) la morphologie du CIV est similaire à myofibroblastes qui sont généralement en forme de fuseau et uniformément répartis sur le flacon. <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Figure 2" /> Figure 2. La morphologie des cellules isolées à la confluence. (A) VEC présentent une morphologie typique endothéliales qui sont généralement pavées et contacter la croissance inhibée. (B) la morphologie du CIV est similaire à myofibroblastes qui sont généralement en forme de fuseau et le contact de croissance n&#39;est pas inhibée. <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Figure 3" /> Figure 3. Les caractéristiques fonction des cellules isolées 6. (A) VEC sont associés à des niveaux élevés d&#39;absorption de LDL acétylées (rouge). (B) CIV sont associés à de faibles niveaux d&#39;absorption LDL acétylées. <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Figure 4" /> Figure 4. Marqueurs cellulaires de cellules isolées 6. (A) phénotype CVE contribué à une coloration positive pour le facteur Willebrand (vert), bleu (noyaux). (B) phénotype VIC contribué à coloration négative facteur von Willebrand. <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Figure 5" /> Figure 5. Marqueurs cellulaires des cellules isolées. (A) phénotype CVE contribué à coloration négative pour alpha SMA (vert), bleu (noyaux). (B) phénotype VIC contribué à coloration positive pour SMA alpha. <table border="1"><tbody><tr><td> Dissociation agent </td><td> La dissociation Technique </td><td> Cell Collection </td><td> Quantité de cellules </td><td> Cellulaire Pureté </td><td> Contamination </td></tr><tr><td> EDTA (3 mM) sans CaCl 2 </td><td> 5, 20, 60 min. CaCl 2 avant le plus </td><td> 20, 60, 120 min. avant la collecte </td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td> EDTA (6mm) sans CaCl 2 </td><td> 5, 20, 60 min. CaCl 2 avant le plus </td><td> 20, 60, 120 min. avant la collecte </td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td> Trypsine-EDTA (0,5 g / L) </td><td> 5, 10, 15 min. avant la désactivation </td><td> Moyen prélevé immédiatement </td><td> + </td><td> + </td><td> + + </td></tr><tr><td> Collagénase II (300 U / ml) </td><td> 5, 10, 15 min. avant la désactivation </td><td> Moyen prélevé immédiatement </td><td> -, +, + + </td><td> -, + +, + </td><td> + </td></tr><tr><td> Collagénase II (600 U / ml) </td><td> 5, 10, 15 min. avant la désactivation </td><td> Moyen prélevé immédiatement </td><td> +, + +, + + + </td><td> + +, + +, - </td><td> - </td></tr></tbody></table> Tableau 1. Résultats préliminaires d&#39;une valvulopathie endothéliales protocoles d&#39;isolement cellulaire.

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

Une bonne compréhension de la biologie valvulaires a été altérée par des difficultés techniques d&#39;isolement et de culture des populations pures de cellules endothéliales valvulaires. Techniques d&#39;isolement typiques impliquent la digestion enzymatique de la matrice sous-jacente basale ou chimique de dissociation des liaisons adhésives endothéliales 2,3. Expériences d&#39;isolement préliminaires ont été évalués qualitativement par la variation des agents de dissociation et de périodes d&...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
<tr>
<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
<tr>
<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
<tr>
<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
<tr>
<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
<tr>
<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25384-092</td>
<td>&nbsp;</td>
<tr>
<td>60x15 Glass Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-310</td>
<td>&nbsp;</td>
<tr>
<td>Paraffin Embedding Wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
<td>&nbsp;</td>
<tr>
<td>Precision Glide Needles</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>305165</td>
<td>&nbsp;</td>
<tr>
<td>500 mL Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-985</td>
<td>&nbsp;</td>
<tr>
<td>1L Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-986</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11023-15</td>
<td>&nbsp;</td>
<tr>
<td>FSC Tweezers #5</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of valvular endothelial cells

Les valves cardiaques sont seul responsable du maintien du flux sanguin unidirectionnel à travers le système cardio-vasculaire. Ces minces, des tissus fibreux sont soumis à des contraintes mécaniques importantes car elles s&#39;ouvrent et se ferment plusieurs milliards de fois sur une durée de vie. L&#39;endurance incroyable de ces tissus est due à la valvulopathie résidents endothéliales (VEC) et les cellules interstitielles (CIV) qui ne cessent de réparation et de remodelage en réponse à des signaux locaux mécaniques et biologiques. Ce n&#39;est que récemment que nous avons commencé à comprendre les comportements uniques de ces cellules, dont l&#39;expérimentation in vitro a joué un rôle clé. Particulièrement difficile est l&#39;isolement et la culture du CVE. Une attention particulière doit être utilisé à partir du moment où le tissu est retiré de l&#39;hôte par plaquage finale. Nous présentons ici des protocoles pour l&#39;isolement direct, l&#39;isolement secondaires spécifiques, la culture, et la vérification des populations pures de CVE. Nous utilisons la digestion enzymatique suivie d&#39;une technique de raclage tige doux pour déloger les cellules de surface seulement. Ces cellules sont ensuite recueillies dans un tube et centrifugée dans une pastille. Le culot est ensuite remis en suspension et plaquées dans des flacons de culture pré-enduites de collagène I matrice. Phénotype VEC est confirmée par le contact a inhibé la croissance et l&#39;expression des marqueurs endothéliaux spécifiques tels que PECAM1 (CD31), facteur von Willebrand (VWF), et l&#39;expression négative de l&#39;actine musculaire lisse alpha (α-SMA). Les caractéristiques fonctionnelles de VEC sont associés à des niveaux élevés de LDL acétylées. Contrairement cellules endothéliales vasculaires, CVE ont la capacité unique de se transformer en mésenchyme, qui se produit normalement lors de la formation embryonnaire, la vanne 1. Cela peut également survenir lors de façon significative après confluentes prolongée de culture in vitro, ainsi le soin doit être fait pour le passage ou à proximité de confluence. Après isolement CVE, des populations pures de CIV peuvent ensuite être facilement acquis.

Watch this Scientific Journal Video about Isolation of Valvular Endothelial Cells at JoVE.com

Isolation of Valvular Endothelial Cells

Nous fournissons une méthode pour isoler et cultiver des populations pures de cellules endothéliales valvulaires cardiaques (VEC). VEC peuvent être isolées de chaque côté de la pointe ou un dépliant et immédiatement après, sous-jacente des cellules interstitielles (CIV) isolement est simple.

L&#39;isolement des cellules endothéliales valvulaires

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

20.3K vues.  Cornell University. Nous fournissons une méthode pour isoler et cultiver des populations pures de cellules endothéliales valvulaires cardiaques (VEC). VEC peuvent être isolées de chaque côté de la pointe ou un dépliant et immédiatement après, sous-jacente des cellules interstitielles (CIV) isolement est simple.

Vidéo: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolement de cellules humaines endotheliales de veine ombilicale (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Recherche

Biologie

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

L&#39;isolement et expansion à grande échelle des cellules adultes humaines Colony Forming progénitrices endothéliales

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolement et culture de cellules progénitrices embryonnaires aviaires valvulaire

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolement et culture de cellules endothéliales pulmonaires des souris néonatales

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolement des cellules endothéliales embryonnaires ventriculaires

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

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolation de microvasculaire endothéliale des artères de résistance Tubes souris

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

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.

Isolement des cellules endothéliales murines Valve

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolement de cellules de souris coronaire endothéliales

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolement et culture des cellules endothéliales aortiques primaires des porcs miniatures

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

L'isolement des cellules endothéliales valvulaires

Résumé

Explorer plus de vidéos

Chapitres dans cette vidéo