ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

Le protocole suivant est effectué conformément aux lignes directrices établies par le ministère de médecine de laboratoire vétérinaire de l&#39;Université de Caroline du Nord à Chapel Hill. 1. Préparer le matériel suivant et réactifs Avant de partir <ol><li> Préparer les médias CE en la complétant 400 ml faible taux de glucose (1 g / L D-glucose ou LG) modifié par Dulbecco du milieu de Eagle (DMEM) avec 50 ml de sérum bovin foetal inactivé par la chaleur, 50 ml Nu-Sérum IV, 5 ml antibiotique-antimycosique, et le hFGF, le VEGF, le hEGF, R3-IGF-1, et des composants de l&#39;héparine à partir du kit commercial. </li><li> Préparer 500 ml FACS tampon (0,5% de BSA et 2 mM d&#39;EDTA dans du PBS); filtrer à travers un filtre de 0,22 um tasse stérile. </li><li> Stériliser ou désinfecter conseil dissection. </li><li> Stériliser les repères de dissection, ciseaux chirurgicaux, et dissecteurs. </li></ol> 2. Isolement CE (Jour 1, ~ 5 h) <ol><li> Euthanasier la souris avec du dioxyde de carbone ou d&#39;autres méthodes d&#39;esprit conformeh Comité des soins et de l&#39;utilisation des animaux institutionnel (IACUC) politiques. Remarque: tumeurs mammaires multiples de taille variable (5 - 15 mm de diamètre) peuvent se développer en une seule souris génétiquement modifiée comme C3-Tag, assurez-vous de les récolter tous pour l&#39;isolement du TEC. Si vous utilisez des tumeurs qui sont orthotopiquement greffées chez la souris, la piscine deux à trois 1 cm 3 tumeurs pour un seul isolement CE. Ici, nous utilisons des tumeurs mammaires comme une démonstration, mais le protocole peut être modifié pour d&#39;autres types de tumeurs. </li><li> Désinfecter souris par pulvérisation ou essuyage de la face ventrale de souris avec une quantité abondante de 75% v / v d&#39;éthanol. </li><li> Réséquer tumeurs avec une paire de ciseaux et ciseaux à l&#39;aide de techniques aseptiques sous une hotte stérile ou à un banc propre. <ol><li> Allongez-vous et épinglez les membres de la souris sur une planche de dissection. Faire une incision médiane ventrale avec les ciseaux sans ouvrir le péritoine. Disséquer latéralement entre la peau et le péritoine vers les glandes mammaires où se trouvent les tumeurs.Ne pas ouvrir le péritoine. </li><li> Accise uniquement de tissu tumoral à partir des glandes mammaires, en laissant de côté les marges mammaires normales. Soigneusement couper les tissus non tumoraux tels que la peau et les muscles, et de placer les tumeurs disséquées dans un tube conique contenant 30 ml de DMEM-LG sur la glace. </li></ol></li><li> Apportez des échantillons de tumeurs à la culture de tissus hotte; laver avec les tissus stériles LG-DMEM 1 - 2 fois. </li><li> Tumeurs de transfert du tube conique à une boîte de Pétri de culture de tissu stérile, ajouter 2 ml de DMEM-LG dans le plat, et mâchent avec une paire de ciseaux stériles en morceaux &lt;5 mm. </li><li> Ajouter 5 ml de la collagénase (Stock = 2 mg / ml dans une solution saline équilibrée de Hank, ci-après HBSS), 1 ml de dispase (stocks = 2,5 U / ml dans HBSS) et 75 ul de désoxyribonucléase (Stock = 1 mg / ml dans PBS ) dans la boîte de Pétri. Le volume total est maintenant ~ 10 ml. </li><li> Transférer le mélange collagénase / tissu de la boîte de Pétri à un tube de tissu-dissociateur et courir sur un dissociateur de tissu pendant 60 secondes deux fois (pré-réglé dissociation prOgram sur le dissociateur: 1.270 tours au total par cycle). Incuber avec la lumière secouant sur un agitateur pendant 75 min à 37 ° C. </li><li> Filtrer à travers un tissu digéré cellule tamis 100 pm sur un tube conique de 50 ml. Le rinçage du filtre avec 5 ml de tampon FACS pour laver toutes les cellules restantes. Spin à 280 g pendant 5 min et aspirer soigneusement le surnageant sans déranger le culot cellulaire. </li><li> Diluer 1 ml d&#39;actions RBC tampon de lyse (10x) dans 9 ml d&#39;eau stérile. Lyse des globules rouges avec 10 ml de tampon de lyse (1x), et immédiatement tourner 5 min à 280 x g. Remarque: Cette étape peut être ignorée si peu de sang est visible. </li><li> Remettre en suspension dans 10 ml de tampon FACS. Mélanger 10 pi de suspension cellulaire avec 10 pi de bleu trypan, et compter les cellules vivantes en utilisant un hémocytomètre. </li><li> Reprendre cellules à ~ 10 7 cellules / 100 pi. Ajouter 10 ul de bloc FcR pour 100 ul de suspension de cellules, et on incube sur de la glace pendant 10 min. </li><li> Ajouter anticorps CD31 de rat anti-souris conjugué à PE selon. Tableau 1 incuber sur de la glace pendant 10 - 15 min et le tube de film de temps en temps. </li><li> Ajouter 10 ml de tampon FACS pour le tube et centrifuger à 280 g pendant 5 min. Retirer délicatement le surnageant et laver le culot cellulaire à nouveau avec 5 ml de tampon FACS. Centrifuger pour culotter les cellules. Aspirer le surnageant sans culot cellulaire inquiétante. </li><li> Ajouter tampon FACS et des microbilles anti-PE conformément au Tableau 2 incuber sur de la glace pendant 10 -. 15 min. Tube de Flick occasionnellement. </li><li> Ajouter 10 ml de tampon et des échantillons de rotation FACS à 280 xg pendant 5 min; laver une fois avec 5 ml de tampon FACS et centrifuger à nouveau. Aspirer le surnageant sans culot inquiétante. </li><li> Amener le volume à 300 pi dans du tampon FACS. Spin à 35 um cellule crépine plafonné tube 5 min à 280 x g. Utilisez 2 tubes et un plus grand volume si nécessaire. </li><li> Mettre en place Multistand magnétique et colonnes magnétiques dans le capot, joindre une colonne pour le séparateur et équilibrer la colonne avec 2 ml de tampon FACS. </li><li> Aspirate le surnageant et remettre le culot cellulaire dans 0,5 - 1 ml de tampon FACS. </li><li> Passez la suspension de cellules à travers la colonne magnétique équilibrée. </li><li> Laver la colonne trois fois avec 2 ml de tampon FACS, et recueillir écoulement (FT) dans un tube de 15 ml (fraction FT). </li><li> Prendre la colonne hors du séparateur et on élue avec 2 ml de tampon FACS dans un autre tube de 15 ml (fraction d&#39;éluat). Utilisez piston pour assurer que toutes les cellules sont de la colonne. Répéter l&#39;élution deux fois avec 2 ml de tampon FACS à chaque fois. </li><li> Spin l&#39;éluat à 280 g pendant 5 min. </li><li> Retirer le surnageant et remettre le culot dans 10 ml de médias CE. </li><li> Tout aussi diviser la fraction d&#39;éluat (~ 6 ml) dans des boîtes revêtues de gélatine 10 cm. Pour trois de 1 cm 3 tumeurs, plaque cellules élue dans au moins quatre plaques. Cellules variante, la plaque est éluée à des concentrations différentes dans plusieurs plaques (par ex., 0,5 ml, ensemencer 1 ml, 1,5 ml et 3 ml de l&#39;éluat en quatre plaques) veiller à ce qu&#39;unt moins une plaque est peu ensemencé avec des cellules élues. Vérifiez que la confluence des cellules attachées est à ~ 1% le lendemain (soit environ 1,0 - 2,0 x 10 5 cellules attachées). Remarque: Les cellules doivent être plaqué rares de sorte que les colonies CE peuvent former sans être contaminés par d&#39;autres types de cellules. </li><li> Plate fraction FT dans un plat de 10 cm pour laisser les cellules récupérer O / N. Vérifiez que la plaque est 80 - 100% de confluence le lendemain. Congeler bas les cellules (-80 ° C) dans 250 médias congélation de cellules-ul dans un cryotube et rangez-les dans de l&#39;azote liquide le lendemain. Remarque: fraction FT peut être utilisé pour l&#39;isolement de cellules de tumeur à un stade ultérieur et / ou en tant que témoin négatif pour l&#39;analyse de l&#39;expression des gènes clones CE CE de isolées. </li><li> Changer de support tous les 2 - 3 jours. Colonies commencent à se former après 7 - 10 jours. Petites colonies CE peuvent être identifiés dès le jour 3. Marquez les colonies avec un marqueur à pointe fine sur le fond du plat. </li><li> Grattez c non spécifiqueaunes entourant les colonies identifiées avec une pointe de Pippet 200 pi stérile. </li></ol> 3. Colony sélection Anneaux Utilisation de clonage <ol><li> Changer de support tous les 2 - 3 jours. CE pureté peut être vérifiée par les LDL (Dil-Ac-LDL) plus à environ 5 - 7 jours. Ajouter 50 pi de LDL par 10 ml de milieu CE et incuber pendant 3 - 4 heures avant de vérifier les cellules sous un microscope à fluorescence. Remarque: LDL Multiple + clones CE pourraient être observés à ~ 7 jours. </li><li> Lancer la récolte des colonies CE quand ils atteignent un diamètre de 3 - 5 mm. (Choisissez grandes colonies qui sont emballés avec les petites LDL + cellules pour de meilleurs résultats.) </li><li> Avant la récolte CE avec des anneaux de clonage, de pré-couche de quelques plaques de 6 puits avec 0,5% de gélatine. Aspirer la gélatine, ajouter 2 ml de milieu CE à chaque puits, et de garder les plaques dans un incubateur jusqu&#39;à ce que nécessaire. </li><li> Grattez non-CE sur les bords des colonies pour vous assurer que pas d&#39;autres types de cellules seront piégés dans le cycle de clonage. </li><li> Utilisation d&#39;unmicroscope à contraste de phase (4X ou 10X objectif), contour avec un marqueur à pointe fine sur le fond de la boîte de culture les zones contenant des colonies de la CE. </li><li> Laver la plaque avec 10 ml de PBS et de laisser une très fine couche de PBS dans la plaque lors de l&#39;aspiration. (Important: une petite quantité (~ 0,5 ml) de PBS va garder les cellules vivantes au cours de la procédure de clonage torique; aussi, tissu adhésif a besoin d&#39;eau pour coller.) </li><li> Choisissez un anneau de clonage de taille appropriée. Utilisez une paire de pince à disséquer pour ramasser une bague, et avec une pipette de 10 pi appliquer uniformément une petite quantité de tissu adhésif sur l&#39;anneau de clonage. Remarque: Utilisez uniquement quantité minimale (~ 0,2 pi pour un petit anneau) d&#39;adhésif de tissu sur des anneaux de clonage, et assurez-vous colle tissulaire est répartie uniformément autour de la surface de fond pour assurer une bonne étanchéité. Adhésif tissulaire excessive produit de la chaleur et fait des films qui peuvent tuer les cellules. </li><li> Placez l&#39;anneau de clonage sur la colonie CE. Appuyez doucement l&#39;anneau de clonage pour coller les ring sur la plaque. Veiller à ce que les colonies ne sont pas séchés avant le collage de la bague. </li><li> Immédiatement pipette 25 pi de solution enzymatique de détachement cellulaire dans l&#39;anneau de clonage et incuber ~ 1 min ou jusqu&#39;à ce que les cellules sont mal fixés. </li><li> Pipet cellules dans l&#39;anneau de clonage goutte à goutte dans un puits d&#39;une plaque à 6 puits contenant les médias communautaires préchauffées. (Important: Ne secouez pas la plaque pour disperser les cellules; CE préfèrent poussent en grappes serrées.) Lavez la bague de clonage avec 50 - 100 médias communautaires ul de collecter autant de cellules que possible, et de transférer tous les lavages dans le même 6 puits . </li><li> Si certaines colonies dans la boîte de 10 cm sont trop petits pour récolter, ajouter 10 ml de milieu frais, laisser les colonies pousser pendant quelques jours de plus, et de répéter la procédure d&#39;anneau de clonage. </li><li> Poussent des colonies récoltées dans des plaques 6 puits jusqu&#39;à 80 - 100% de confluence, et le transfert des cellules à 3 puits d&#39;une plaque à 6 puits, avant de les étendre dans une boîte de culture de tissu de 10 cm. Grattez cellules contaminantes. Répéterune autre série de la procédure de l&#39;anneau de clonage (étapes 03/05 au 03/10), si nécessaire. Gardez cellules relativement confluentes (~ 60 - 70%) lors de l&#39;expansion. CE peut cesser de croître si plaqué trop clairsemée. </li><li> Caractériser CE par FACS, la coloration, PCR, etc. Notez que Dil-Ac-LDL est fluorescent et peut interférer avec PE ou d&#39;autres anticorps fluorescents pour FACS. </li></ol>

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Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, 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=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

En raison des difficultés à obtenir des cultures de TEC primaires pures, dont beaucoup dans des études in vitro TEC substitut avec des lignes disponibles dans le commerce de la CE ou primaire CE tels que la veine ombilicale humaine CE (HUVEC) 13. Cependant, ces populations CE de tissus normaux ne peuvent servir de proxy pour les TEC qui diffèrent sensiblement de leurs homologues normales. Par exemple, TEC sont phénotypiquement et fonctionnellement anormale in vivo et certains de ces ano...

Les cellules endothéliales (CE) sont essentiels lors du développement de tumeurs solides. De l&#39;initiation du switch angiogénique dans les tumeurs dormantes à la diffusion et l&#39;ensemencement des métastases à des sites distants, CE former les conduits qui fournissent du sang, de l&#39;oxygène et de nutriments pour soutenir la croissance de la tumeur 1. Comme suggéré récemment, EC ont aussi des fonctions de perfusion indépendantes et former une niche qui prend en charge la croissance des cellules souches cancéreuses et d&#39;autres cellules du stroma de la tumeur 2-5. Ainsi, hautement purifié CE (TCE) pour la culture in vitro spécifique de la tumeur permet études fonctionnelles de routine qui va faire la lumière sur les mécanismes moléculaires de médiation angiogenèse tumorale et la diaphonie avec des cellules tumorales. CE sont très spécialisés en fonction du tissu d&#39;origine 6. En raison de la nature hétérogène de différents types de tumeurs et le microenvironnement de la tumeur, TEC peut également afficher des caractéristiques uniques qui reflètent une tumeur spécifique à la spécialisation of le système vasculaire. Par exemple, il existe une variabilité frappante dans les signatures d&#39;expression génique dans TEC isolés à partir de différents types ou classes de tumeurs 7,8. Toutefois, fréquemment co-purification de non-CE, en particulier les fibroblastes associés à une tumeur et les cellules tumorales, avec TEC peut confondre analyses d&#39;expression du génome entier. Ces types de cellules non désirées sont particulièrement problématiques dans les études qui reposent sur ​​le long terme dans l&#39;expansion in vitro de cultures de TEC. Décrit ici est un procédé haute-fidélité qui produit toujours des cultures pures CE de tumeurs et d&#39;autres tissus. Suite à l&#39;enrichissement immunomagnétique des fractions de la CE et l&#39;élimination des co-purifié non-CE colonne, une étape clonage torique supplémentaire pour capturer colonies CE pur est utilisé 9. Chaque colonie peut être étendue en culture pendant plusieurs passages sans l&#39;émergence de contamination non-CE. Cette méthode donne également de multiples clones CE d&#39;une procédure d&#39;isolement unique, ce qui est idéal pour l&#39;étude de l&#39;endohétérogénéité épithélial. En outre, il est démontré que Cdh5 CRE: les souris rapporteurs / l de ZsGreen l / sont un outil précieux pour générer CE &quot;de sort-mappé&quot; et indélébile marqué qui maintiennent ZsGreen fluorescence dans la culture 10. Avec quelques ajustements mineurs au protocole, cette méthode devrait être adaptable à différents types de tumeurs ou de tissus normaux.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

Fraîchement isolés cellules endothéliales spécifiques de tumeurs (TEC) peuvent être utilisées pour étudier les mécanismes moléculaires de l&#39;angiogenèse tumorale et servir comme un modèle in vitro pour le développement de nouveaux inhibiteurs de l&#39;angiogenèse pour le cancer. Cependant, à long terme dans l&#39;expansion in vitro des cellules endothéliales murines (CE) est difficile en raison de la dérive phénotypique dans la culture (de transition endothéliale-à-mésenchymateuses) et la contamination avec des non-CE. Cela est particulièrement vrai pour TEC qui sont facilement surpassés par les fibroblastes co-purifié ou des cellules tumorales en culture. Ici, une méthode d&#39;isolement de haute fidélité qui tire parti de l&#39;enrichissement immunomagnétique couplée avec la sélection de la colonie et dans l&#39;expansion in vitro est décrite. Cette approche génère des fractions pures CE qui sont entièrement libres de contaminer les cellules stromales ou tumorales. Il est également démontré que la lignée tracée Cdh5-cre: / l de souris rapporteurs de ZsGreen l /, utilisés avec le protocole décrits ici, sont un outil précieux pour vérifier cellulairela pureté que les colonies isolées CE de ces souris montrent durable et brillante fluorescence ZsGreen dans la culture.

CE représente seulement une fraction mineure de la population cellulaire totale dans la plupart des tissus adultes 11. Il est donc important pour digérer complètement le tissu récolté dans une suspension à cellule unique qui assure la libération maximale de CE à partir de la matrice extracellulaire (ECM) et les tissus conjonctifs. Dans notre expérience, la sélection immunomagnétique CD31 à médiation ne fournit fractions CE enrichis mais pas pures; par conséquent, une autre étape cruciale est l&...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolement et culture Expansion des cellules endothéliales spécifique de la tumeur

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

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JoVE Journal

Medicine

11.1K vues.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Vidéo: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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Médecine

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

L&#39;isolement, la culture, et de l&#39;imagerie de l&#39;homme causés par des amas de cellules pancréatiques

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolement, cryoconservation et de la culture des droits de cellules épithéliales Amnion pour des applications cliniques

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolement des cellules endothéliales humaines lymphatique par multi-paramètres fluorescence tri cellulaire activé

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolement et culture de cellules endothéliales primaires de Artères Canine et Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Isolement de cellules progénitrices endothéliales de volontaires sains et de leur potentiel migratoire Influencé par échantillons de sérum après chirurgie cardiaque

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

préparation et<em&gt; In vitro</em&gt; Caractérisation des aimanté miR-modifié cellules endothéliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Évaluation des propriétés anticoagulantes et anti-inflammatoires de cellules endothéliales à l’aide de la Culture cellulaire 3D et sang total Non-anticoagulant

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Les déséquilibres musculaires : Essais et formation fonctionnelle ischio-jambiers excentrique force athlétique populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...