Cette recherche a été financée en partie par l&#39;American Heart Association 0950118G.to octroi MC-W.

1. Préparation anti-PECAM-1-anticorps conjugués des billes magnétiques (Dynabeads) <ol><li> Préparer 0,1% de BSA dans du PBS en mélangeant 50 mg de BSA dans 50 ml de PBS en utilisant un vortex jusqu&#39;à BSA dissout. Stériliser par filtration sur filtre de 0,22 um seringue. Conserver à 4 ° C. </li><li> Dans la hotte de TC, le transfert de 200 pi de bien resuspendu Dynabeads IgG de mouton anti-rat dans un tube Eppendorf de 1,5 ml et laver les perles: tube mont sur le MPC et laisser reposer pendant environ 1 min pour permettre aux billes de sédiments. Aspirer le surnageant, retirer le tube du MPC et de remettre en suspension les perles dans 1 ml de solution stérile de 0,1% BSA / PBS. Pipeter haut et bas pour resuspendre perles. </li><li> Répétez laver plus de trois fois, pour un total de quatre lavages. </li><li> Retirer le tube du MPC et de remettre en suspension les perles dans 500 ul de 0,1% de BSA / PBS. </li><li> Ajouter 10 ul rat anti-souris PECAM-1 (CD-31) anticorps pour le tube. </li><li> Tumble nuit à 4 ° C dans la chambre froide ou 2 heures à température ambiante. </li><li> Laver les billes avec stériles 0,1% BSA / PBS comme décrit dans l&#39;étape 1.2 à quatre reprises. </li><li> Retirer le tube du MPC et de remettre en suspension les perles dans 200 ul de 0,1% BSA / PBS. Boutique anti-PECAM-1-anticorps conjugués Dynabeads à 4 ° C pour un maximum de 1 mois. </li></ol> 2. Isoler la souris cellules endothéliales pulmonaires chez les souris néonatales Avant de procéder avec le protocole de purification des tissus, IACUC approbation du Comité de la procédure est nécessaire. <ol><li> Préparer les éléments suivants: <ul><li> 15 ml de 1 mg / ml C / D solution dans du DMEM. Filtre avec une seringue 0,22 um filtre et chaude à 37 ° C. </li><li> Tube de 50 ml conique avec 15 ml de DMEM, de stocker sur la glace </li><li> Isolement des médias (IM) contenant 20% de FBS et 1x pénicilline / streptomycine dans le DMEM </li><li> 2% de gélatine. Mélanger 2 g de gélatine bovine peau avec 100 ml d&#39;eau dd, autoclave 15 minutes et conserver à 4 ° C. Chaude à 37 ° C pour liquéfier avant flacons TC revêtement. </li></ul></li><li> Anesthetize trois chiots 6-8 jours anciens en utilisant une injection IP de kétamine (100 mg / kg) et de xylazine (10 mg / kg). Vérifier l&#39;efficacité de l&#39;anesthésie et disséquer des animaux, un par un, comme suit: chiots Pin à bord, faire tremper la peau avec de l&#39;éthanol 70% et enlever la peau de la région de la poitrine avec des ciseaux de dissection stériles. Accise de la cage thoracique afin d&#39;exposer les poumons. </li><li> Aseptiquement accise lobes pulmonaires individuelles, en faisant attention de ne pas disséquer les bronches et les tissus conjonctifs visibles autour des poumons. Combiner tous les poumons glacée DMEM. Euthanasier les chiots. </li><li> Travailler dans la hotte TC, retirez les lobes pulmonaires du DMEM, placer dans un plat de 100 mm TC et aspirer l&#39;excès DMEM. </li><li> L&#39;aide de ciseaux stériles, émincer finement le tissu en coupant ~ 100 fois. Transfert à 15 ml chaleureuse C / D pour la solution de digestion enzymatique. Incuber 45 minutes à 37 ° C sur un agitateur. </li><li> Dans la hotte TC, aspirer la suspension de tissu digéré en une seringue de 20 ml avec 14 g de touffes canule jointe et triturer dans une suspension cellulaire unique, au moins 12 fois. </li><li> Passez la suspension de tissu à travers un tamis 70 pm cellules et laver le filtre avec 15 ml IM à arrêter la digestion. </li><li> Suspension cellulaire par centrifugation à 400x g pendant 5 minutes. </li><li> Aspirer le surnageant et remettre le culot dans 3 ml de 0,1% BSA / PBS. </li><li> Transfert suspension cellulaire à 5 ml tube de polystyrène à fond rond et ajouter 22,5 ul anti-PECAM-1-anticorps conjugués Dynabeads. Tumble à température ambiante pendant 12 minutes. </li><li> Manteau un flacon T75 avec 2 mL 2% de gélatine et de l&#39;excès d&#39;aspiration de la gélatine. Laisser sécher la gélatine à l&#39;intérieur du capot de TC pendant environ 30 minutes avant de cellules de placage. </li><li> Après une incubation de perles est terminé (étape 2.10), répartis de façon égale en suspension de cellules des tubes Eppendorf trois et monter sur la MPC. </li><li> Lorsque les billes ont sédimentées en utilisant le MPC (~ 1 min), ramasser le surnageant, retirer le tube du MPC, lavez perles avec ~ 1 ml de 0,1% BSA / PBS, puis remontez sur la MPC. </li><li> Répétez laver quatre fois (au total 5 lavages) </li><li> Perles Remettre en suspension dans 1 ml de VascuLife avec des facteurs de vie EnGS-MV Kit et 1x pénicilline / streptomycine (VL) par tube, bien mélanger. </li><li> Plaque sur un pré-enduites flacon T75 et amener le volume total dans chaque flacon de 10 ml avec VL. </li><li> Changer les médias le lendemain, permettra un jour complet de la croissance, puis le changement de la moitié des médias tous les jours. Lorsque les cellules sont&gt; 70-80% confluentes ou plus (généralement après 3-4 jours de culture), ils peuvent être triés avec des anti-ICAM-2 Dynabeads. </li></ol> 3. Préparation anti-ICAM-2 anticorps conjugués Dynabeads <ol><li> Dynabeads anti-ICAM-2 anticorps conjugués sont utilisés pour purifier davantage les cellules qui ont été sélectionnés avec des anti-PECAM-1-anticorps conjugués Dynabeads et cultivée jusqu&#39;au confluent. Cette procédure est réalisée comme décrit pour Dynabeads anti-PECAM-1-anticorps conjugués (1, ci-dessus), sauf que l&#39;anti-souris ICAM-2 (CD-102) anticorps est utilisé à la place de l&#39;anti-PECAM-1 anticorps. </li><li> Dynabeads anti-ICAM-2 anticorps conjugués peuvent être conservés à 4 ° C jusqu&#39;àà 1 mois. </li></ol> 4. Le tri des cellules de poumon de la souris endothéliales avec anti-ICAM-2 Dynabeads <ol><li> Manteau T75 flacon de culture tissulaire avec 2% de gélatine. </li><li> Aspirer les médias de la confluence flacon T75 avec des cellules et laver avec 8 ml de PBS. </li><li> Aspirer le PBS, ajouter 2 ml 0,05% de trypsine / EDTA et laissez cellules se détachent (environ 5 minutes). Tap flacons légèrement pour détacher l&#39;aide. </li><li> Lorsque les cellules se sont détachées, ajouter 2 ml IM à inhiber la trypsine les cellules et gratter pour détacher les cellules adhérentes restantes. Transfert suspension cellulaire dans un tube de 15 ml et centrifuger pendant 5 min à 400x g. </li><li> Aspirer les médias et les cellules Resuspendre le culot dans 2 ml de 0,1% BSA / PBS. </li><li> Transférer dans un 5 ml en polystyrène à fond rond tube et ajouter 10 ul anti-ICAM-2 Dynabeads. Tumble pendant 12 minutes à température ambiante. </li><li> Fractionner cellules en suspension dans deux tubes de 1,5 ml Eppendorf et monter sur le MPC. </li><li> Aspirer le surnageant après perles ont adhéré pendant une minute. Retirer les tubes de MPC et resuspendre chaque tube dans 1 ml de 0,1% BSA / PBS. Remontez sur le MPC. </li><li> Répétez laver quatre fois (total de 5 lavages). </li><li> Resuspendre chaque tube avec 1 ml VL et d&#39;effectuer un comptage des cellules. </li><li> Cellules de la plaque à 250-350K cellules par flacon T25 et / ou 750K-1 millions de cellules par flacon T75. </li><li> Amener le volume à 5 ml VL dans des flacons T25 et T75 10 ml dans des flacons. </li><li> Changer les médias tous les jours. Les cellules peuvent être utilisés pour des expériences lorsque la culture atteint environ 80% de confluence, généralement en 2-3 jours. </li></ol> 5. Les résultats représentatifs Généralement, après 6-7 jours de la préparation initiale des cellules, nous sommes en mesure d&#39;obtenir environ 01.02 à 01.05 x 10 6 cellules endothéliales pulmonaires de trois chiots 6-8 jours anciens. Cellules d&#39;affichage typique »pavées&quot; morphologie en microscopie optique et de montrer la VE-cadhérine (CD144) à la coloration jonctions cellule-cellule, qui est caractéristique des cellules endothéliales (figure 1). La majorité des MLECs expresse VE-cadhérine et VEGFR2 comme démontré par analyse FACS (figure 2). Habituellement, nous les utilisons pour des expériences dans les deux semaines après l&#39;isolement initial MLEC. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="Figure 1" /> Figure 1. L&#39;analyse microscopique des MLEC monocouche confluente. (A) Image au microscope optique montre la morphologie des cellules cultivées pavées typiques des cellules endothéliales. Uniforme structures rondes situées dans la région périnucléaire de nombreuses cellules sont les billes magnétiques utilisées pour l&#39;isolement MLEC. (B) endothélial spécifique VE-cadhérine est localisée au niveau des jonctions cellule-cellule comme indiqué en utilisant la microscopie confocale. Bar est représentatif de 20μm. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="Figure 2" /> Figure 2 analyse FACS des MLECs culture marquées avec des anticorps spécifiques pour endothéliales des marqueurs spécifiques:. VE-cadhérine (CD144) ou VEGFR2, comme inculpé, suivie par la phycoérythrine anticorps secondaire conjugué, confirme l&#39;identité de l&#39;endothélium MLECs isolés. La ligne rouge indique isotype-spécifiques de contrôle, la ligne verte indique anti-VE-cadhérine ou anti-VEGFR2 - spécifiques IgG.

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. G., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+vascular+endothelial+cadherin+(7B4/cadherin-5),+an+endothelium-specific+cadherin.">Functional properties of human vascular endothelial cadherin (7B4/cadherin-5), an endothelium-specific cadherin.</a> Arterioscler Thromb Vasc Biol. 15, 1229-1239 (1995).</li><li>Zachary, I., Gliki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+transduction+mechanisms+mediating+biological+actions+of+the+vascular+endothelial+growth+factor+family.">Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family.</a> Cardiovasc Res. 49, 568-581 (2001).</li><li>Diglio, C. A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. 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Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements stipulés par Medical College of Wisconsin IACUC Comité en vertu du protocole AUA # 00001206.

Microvasculaire EC se sont révélés être un modèle utile dans de nombreux domaines de la biologie vasculaire et sont considérés comme physiologiquement plus pertinent d&#39;étudier (par exemple l&#39;angiogenèse) que largement étudié HUVEC 3. Auparavant, il a été rapporté que microvasculaires ECS peut être obtenu à partir de rein, cœur, peau de tissu adipeux, la rétine, le cerveau, le gliome, et aussi des poumons 2, 4-7, 10, 11. Toutefois, une méthode cohérente et fiable pour l&#39;isolement des complications microvasculaires ECS est toujours requis. La procédure présentée ici est une modification d&#39;un protocole de deux publiés antérieurement; elle diffère de la plupart des procédures publiées en ce qu&#39;elle utilise les chiots au lieu d&#39;animaux adultes. Ceci est crucial pour le succès d&#39;isolement que les cellules de jeunes animaux ont un potentiel élevé de prolifération et de cultures dérivées de leurs tissus ont tendance à donner plus grand nombre de cellules. Une semaine anciens animaux semblent être optimales mais les jeunes souris (quatre jours d&#39;âge) peuvent également être utilisés. En outre, nombre supérieur ou inférieur de chiots peut être utilisée si nécessaire, comme nous avons réussi à isoler MLECs d&#39;un chiot. Cependant, tout en réduisant le haut ou vers le bas le processus d&#39;isolement, densité de placage cellulaire doit rester inchangée, comme cela est également critique pour la prolifération cellulaire. Le processus de purification en 2 étapes du MLECs utilisant des billes magnétiques conjuguées premier anti-PECAM-1 et que les anti-ICAM-2 anticorps est beaucoup plus efficace d&#39;obtenir des cultures pures de CE que les procédures décrites précédemment de purification en une seule étape. Ce protocole élimine la nécessité d&#39;utiliser des techniques manuelles très laborieux, la centrifugation de gradient et FACS moins efficace de tri des cellules jeter polluants tels que les fibroblastes, les cellules sanguines et des cellules musculaires lisses. La population résultant MLEC peut être utilisé pour l&#39;analyse in vitro des réponses angiogéniques, la perméabilité vasculaire et la transmigration des leucocytes, la cicatrisation des plaies ainsi que l&#39;analyse biochimique des voies de signalisation. Si nécessaire, MLECs peuvent être plaqués sur différentes surfaces telles que des lamelles de fibronectine ou de la gélatine à revêtement pour les tableaux immunofluorescence ou d&#39;électrodes pour la résistance trans-endothéliale (TER) mesures. Les résultats obtenus peuvent être comparés directement aux phénotypes observés in vivo, ce qui est particulièrement important dans le contexte de l&#39;augmentation du nombre des huitièmes de finale et de lignées de souris transgéniques. Mise en œuvre réussie de cette méthode pour l&#39;analyse in vitro des mécanismes cellulaires qui sous-tendent les défauts observés in vivo, a déjà été présentée 12.

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		<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>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of pulmonary endothelial cells from neonatal mice

Les cellules endothéliales de fournir un modèle de recherche utiles dans de nombreux domaines de la biologie vasculaire. Depuis son premier isolement 1, des cellules endothéliales de veine ombilicale (HUVEC) ont montré pour être pratique, facile à obtenir et à la culture, et sont donc les cellules endothéliales plus largement étudié. Cependant, pour des recherches axées sur les processus de l&#39;angiogenèse comme les autres, la perméabilité ou plusieurs cellules endothéliales microvasculaires (ECs) sont un modèle beaucoup plus physiologiquement pertinents pour l&#39;étude 2. Par ailleurs, EC isolées de souris knockout fournir un outil utile pour l&#39;analyse des protéines ex vivo la fonction. Plusieurs approches d&#39;isoler et de la culture microvasculaires ECs de différentes origines ont été rapportés à ce jour 3-7, mais l&#39;isolement et la culture cohérente de pure ECS est toujours un problème technique majeur dans de nombreux laboratoires. Ici, nous fournissons un protocole étape par étape sur une méthode fiable et relativement simple d&#39;isoler et de cultiver des cellules de souris pulmonaires endothéliales (MLECs). Dans cette approche, les tissus pulmonaires obtenues à partir de 6 - 8 jours chiots ancienne est d&#39;abord coupé en morceaux, digérés par la collagénase / dispase (C / D) en solution et dispersés mécaniquement dans une seule cellule de suspension. MLECS sont purifiés à partir de cellules en suspension en utilisant une sélection positive avec des anti-PECAM-1 anticorps conjugué à Dynabeads utilisant un concentrateur de particules magnétiques (MPC). Ces cellules purifiées sont cultivées sur la culture de tissus enduits de gélatine (TC) plats jusqu&#39;à ce qu&#39;ils deviennent confluentes. A ce stade, les cellules sont encore purifiée en utilisant Dynabeads couplé à l&#39;anti-ICAM-2 anticorps. MLECs obtenus avec ce protocole présentent un phénotype pavées, comme visualisé par microscopie optique à contraste de phase, et leur phénotype endothélial a été confirmée en utilisant une analyse FACS avec des anti-VE-cadhérine 8 et anti-VEGFR2 9 anticorps et coloration par immunofluorescence de la VE-cadhérine. Dans nos mains, cette procédure d&#39;isolement en deux étapes cohérente et fiable des rendements d&#39;une population pure de MLECs, qui peut encore être cultivé. Cette méthode permettra aux chercheurs de profiter du nombre croissant de knock-out et des souris transgéniques pour une corrélation directe entre les études in vivo avec des résultats d&#39;expériences in vitro réalisées sur MLECs isolées et contribuer ainsi à révéler des mécanismes moléculaires des phénotypes vasculaires observés in vivo.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Ici, nous décrivons un protocole pour l&#39;isolement et la culture de cellules endothéliales murines pulmonaires. Cette méthode comprend mécanicien et dissociation enzymatique du tissu pulmonaire ainsi que d&#39;un processus de purification en 2 étapes utilisant des anticorps anti-PECAM-1 et anti-ICAM-2 anticorps conjugués à des billes magnétiques, ce qui produit une population de cellules endothéliales pures d&#39;origine essentiellement microvasculaires.

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 ...

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JoVE Journal

Biology

46.9K vues.  BloodCenter of Wisconsin. Ici, nous décrivons un protocole pour l'isolement et la culture de cellules endothéliales murines pulmonaires. Cette méthode comprend mécanicien et dissociation enzymatique du tissu pulmonaire ainsi que d'un processus de purification en 2 étapes utilisant des anticorps anti-PECAM-1 et anti-ICAM-2 anticorps conjugués à des billes magnétiques, ce qui produit une population de cellules endothéliales pures d'origine essentiellement microvasculaires.

Vidéo: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

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 ...

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Biologie

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolement et culture de cardiomyocytes de souris néonatales

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolement de l&#39;artère pulmonaire cellules musculaires lisses de souris néonatales

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Génération et de la Culture du sang excroissance des cellules endothéliales de sang périphérique humain

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

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 Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...