Vorremmo ringraziare Andrea Portbury per la lettura critica del manoscritto, il microscopio Servizio Laboratorio di UNC per l&#39;assistenza con il time-lapse imaging, e il NIH (borsa # R01HL061656) per il supporto finanziario.

1. Preparare Beads anticorpi coniugati <ol><li> Un giorno prima della dissezione, unire l&#39;anticorpo di scelta con le Dynabeads appropriate (ad esempio ratto IgG Dynabeads per un anticorpo cresciuto in ratto, 4 x 10 8 perline / ml) per le istruzioni del produttore. Per il BD ratto anti-CD31 anticorpo (0,5 mg / mL, per isolare le cellule endoteliali), è aggiunto 3 anticorpo microlitri per ogni 25 microlitri perline, per una concentrazione finale di 1,5 mg anticorpo anti-CD31 e 1 x 10 7 perline / 25 microlitri di buffer. </li><li> Incubare perline e anticorpi notte a 4 ° C. </li></ol> 2. Preparazione del pannello di collagene <ol><li> Cellule isolate formano tubuli più rapidamente quando coltivate su un gel di collagene rispetto al solo una piastra rivestita di collagene. Pertanto, gel di collagene sono fatte la notte prima della dissezione, come descritto al punto 6. Sul ghiaccio in una cappa di coltura tissutale, combinare i seguenti reagenti (sufficiente per 19 pozzetti): 206,4 ml sterile H &lt;sub&gt; 2 O, 140,4 microlitri collagene di tipo I (4,08 mg / ml), 38,4 microlitri 10x M199, e 1,15 microlitri 5 M NaOH. </li><li> Pipettare 20 ml di gel di collagene presente nei pozzetti di una piastra di coltura tissutale a 384 pozzetti. Porre la piastra in una ° C di coltura tissutale incubatore 37 per 30 min. </li><li> Aggiungere 80 microlitri DMEM a ciascun pozzetto e incubare per 15 minuti a 37 ° C. Rimuovere il terreno di coltura e ripetere altre due volte. </li><li> Dopo l&#39;ultimo risciacquo, aggiungere 80 microlitri 10% FBS / DMEM a ciascun pozzetto e incubare una notte a 37 ° C. Rimuovere il mezzo di coltura prima di aggiungere le cellule isolate. </li></ol> 3. Accise e digerire il Cuore <ol><li> Eutanasia di un mouse timed-incinta al giorno embrionale desiderato utilizzando una tecnica di eutanasia approvato. Tutti gli esperimenti sono stati approvati dalla cura degli animali e del Comitato Istituzionale Usa presso la University of North Carolina a Chapel Hill. </li><li> Con il mouse in posizione supina, liberalmente spruzzare il lato ventrale della femmina con il 70% di etanolo primadissezione. Utilizzando pinze per sollevare la pelle addominale inferiore e muscolo, allontanandosi dagli organi interni, tagliare aprire cavità inferiore addominale della femmina di visualizzare gli organi interni. </li><li> Tenendo il collo dell&#39;utero con una pinza, tagliare con cura caudale per la pinza e iniziare il sollevamento del corno uterino dalla cavità addominale. Mentre si solleva il corno uterino, tagliare qualsiasi tessuto connettivo che tiene il corno a posto. Per finire ablazione del corno uterino, tagliare il nodo del corno uterino con l&#39;ovidotto per liberare il corno uterino. </li><li> Collocare il corno uterino in una capsula di Petri contenente freddo 1x tampone fosfato salino (PBS) e sciacquare come necessario. Tagliare il corno uterino attraverso la linea mediana per esporre i sacchi embrionali allegati. </li><li> Tagliare un sacco embrionale all&#39;incrocio tra la placenta e l&#39;embrione di evitare accidentalmente tagliare l&#39;embrione e recuperare un embrione. Posizionare l&#39;embrione in una seconda capsula di Petri con PBS freddo. Riportare la capsula di Petri con il corno uterino di ghiaccio. </li><li> Decapitate l&#39;embrione per migliorare l&#39;accesso alla parete toracica. Se genotipizzazione è necessario, tagliare la coda per rimuoverla e salvare in una provetta Eppendorf posti su ghiaccio. </li><li> Con l&#39;embrione sul dorso, tagliare aprire la parete toracica per visualizzare il cuore e polmoni. Per evitare di tagliare il cuore, fare un taglio verticale lungo il lato della gabbia toracica, vicino a una zampa anteriore, seguito da un taglio orizzontale attraverso la parte inferiore delle costole, poi estrarre delicatamente la parete toracica e fuori del modo. Attraverso circa embrionale giorno 13.5, la parete toracica è trasparente, aiutando nella visualizzazione. </li><li> Sollevare delicatamente il cuore con pinze e tagliare i vasi sotto. Quindi, tagliare sopra i grandi vasi di liberare il cuore. Se i vasi polmonari non sono tagliati, i polmoni possono essere rimossi con il cuore. Così, eliminano tessuti estranei, come i polmoni, se necessario. </li><li> Separare e scartare i vasi atri e grande dai ventricoli. Utilizzando le forbici, tritare i ventricoli in piccoli pezzi (circa 1 mm 3 &lt;/ Sup&gt;) e il luogo in circa 500 ml di collagenasi (1 mg / ml in PBS sterile). Conservare i campioni in ghiaccio fino a quando tutti i ventricoli sono stati macinati e messi in collagenasi. </li><li> Ripetere i passaggi precedenti fino a quando i ventricoli sono stati rimossi da tutti gli embrioni, raccogliendo ogni cuore in un tubo Eppendorf separata. Se tutti gli embrioni hanno lo stesso genotipo, i cuori possono essere raggruppati in un unico tubo Eppendorf. Il numero di campioni trattati in una sola volta può essere limitato in base al magnete utilizzato per l&#39;isolamento, la DynaMag (123-21D), qui usato in possesso di un massimo di 16 provette Eppendorf. </li><li> Posizionare i ventricoli tritate a 37 ° C con dondolo per 45 min. Ogni 15 minuti, rimuovere delicatamente il campione e pipettare su e giù per dissociare meccanicamente le cellule. </li></ol> 4. Isolando le cellule endoteliali <ol><li> Far passare la soluzione ventricolo-collagenasi attraverso un 70 micron filtro per rimuovere macchie residue di cellule. </li><li> Centrifugare le cellule a 400 xg per 10 min. Scartareil surnatante, sostituire con circa 200 ml 10% FBS in DMEM, e pipettare su e giù per dissociare le cellule. </li><li> Ripetere il passaggio precedente una volta. Centrifugare le cellule a 400 xg per 10 min dopo il secondo 10% FBS / DMEM lavaggio. Risospendere le cellule in 50 microlitri 10% FBS / DMEM. </li><li> Durante la fase di centrifugazione finale, preparare le perline. <ol><li> Porre il tubo Eppendorf contenente l&#39;anticorpo e perline su un magnete per 1 min. </li><li> Con il tubo in posizione sul magnete, rimuovere il surnatante. Togliere la provetta dal magnete e aggiungere circa 100 ml 10% FBS / DMEM. </li><li> Posizionare il tubo sul magnete per 1 min. Rimuovere il surnatante, e lavare ancora con 10% FBS / DMEM. Ripetere per un totale di 3 lavaggi. </li><li> Dopo l&#39;ultimo lavaggio, risospendere le sfere di anticorpi coniugati a 10% FBS / DMEM (5 ml per campione). </li></ol></li><li> Per ciascun campione cellulare, aggiungere 5 ml di perline anticorpo-coniugate. Posizionare le sospensioni bead-cell a 4 ° C con dondolo for 30 min. </li><li> In una cappa a flusso laminare, posizionare le provette Eppendorf contenenti cellule e perline anticorpi coniugati sul magnete per 1 min. Eliminare il surnatante, togliere i tubi dal magnete, e lavare con circa 100 l 10% FBS / DMEM. Ripetere l&#39;operazione per 5 lavaggi. </li><li> Dopo l&#39;ultimo lavaggio, posizionare i tubi sul magnete per 1 min, rimuovere il supernatante, e rimuovere i tubi dal magnete. Aggiungere circa 100 microlitri DMEM. </li><li> Porre i tubi sul magnete per 1 min, rimuovere il supernatante, e rimuovere i tubi dal magnete. Aggiungere 100-200 ml preriscaldata 0,25% tripsina-EDTA. Incubare i campioni a 37 ° C e 5% di CO 2 per 5 min. </li><li> Posizionare i tubi sul magnete per 2 min. Trasferire accuratamente il supernatante contenente cella per provette Eppendorf. Centrifugare per 5 min a 400 x g. </li><li> Rimuovere il surnatante. Risospendere le cellule in 80-100 microlitri mezzo di crescita e piatto in un 96 - o 384 pozzetti piatto di coltura trattata, a seconda del rendimento atteso(Circa 450-600 cellule dai ventricoli del E14.5-E16.5 embrioni). Per le cellule endoteliali, utilizzare endoteliale coltura mezzo di crescita (definito di seguito). </li></ol>

<ol>
	<li>Landecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Life: How Cells Became Technologies. , (2007).</li><li>Kan, C. Y., Wen, V. W., 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+dysfunction+and+cytoskeletal+changes+associated+with+repression+of+p16(INK4a)+during+immortalization.">Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16(INK4a) during immortalization.</a> Oncogene. , (2012).</li><li>Dyer, L. A., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+endothelium:+an+emphasis+on+heterogeneity.">Development of the endothelium: an emphasis on heterogeneity.</a> Semin. Thromb. Hemost. 36, 227-235 (2010).</li><li>Dong, Q. G., Bernasconi, 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=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 17, 1599-1604 (1997).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. 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+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3, 1085-1091 (2008).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+a+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.</a> Dev. Biol. 95, 108-114 (1983).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nat. Protoc. 5, 628-635 (2010).</li><li>Zeisberg, E. M., Potenta, S. E., Sugimoto, H., Zeisberg, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+kidney+fibrosis+emerge+via+endothelial-to-mesenchymal+transition.">Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.</a> Journal of the American Society of Nephrology : JASN. 19, 2282-2287 (2008).</li><li>Chang, L., Noseda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+vascular+smooth+muscle+cells+from+local+precursors+during+embryonic+and+adult+arteriogenesis+requires+Notch+signaling.">Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.</a> PNAS. 109, 5993-6998 (2012).</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> American Journal of Physiology: Lung Cellular and Molecular Physiology. 296, L1095-L1103 (2009).</li><li>Frauli, M., Ludwig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+fibroblast+proliferation+in+a+culture+of+human+endometrial+stromal+cells+using+a+medium+containing+D-valine.">Inhibition of fibroblast proliferation in a culture of human endometrial stromal cells using a medium containing D-valine.</a> Archives of Gynecology and Obstetrics. , 241-96 (1987).</li><li>Lazzaro, V. A., Walker, R. 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=Inhibition+of+fibroblast+proliferation+in+L-valine+reduced+selective+media.">Inhibition of fibroblast proliferation in L-valine reduced selective media.</a> Research communications in chemical pathology and pharmacology. 75, 39-48 (1992).</li><li>Arima, S., Nishiyama, K., 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=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Lavorare con le cellule embrionali primarie consente nuovi esperimenti per affrontare in fasi critiche dello sviluppo in vitro. Tuttavia, la procedura di isolamento non è banale. Passaggi critici per isolare qualsiasi tipo di cellule comprendono assicurare che le cellule sono ben separati su digestione collagenasi e poi che siano ben sospesi durante le fasi di lavaggio. Dissezione meccanica pipettando aiuta notevolmente separare le cellule durante la fase di collagenasi, e la fase di filtraggio rimuov...

L&#39;avvento di linee cellulari immortalizzate ha rivoluzionato la biologia cellulare di base 1. Le linee cellulari attualmente disponibili sono derivati ​​da una vasta gamma di organi e comprendono tutti i principali tipi di cellule. Tuttavia, le linee cellulari stabilizzate hanno alcune limitazioni. Il processo di immortalizzazione ovviamente cambia il comportamento delle cellule, in particolare rispetto alla durata di vita e la proliferazione, ma può anche influenzare l&#39;espressione di proteine ​​imprevisti, come le proteine ​​citoscheletriche 2. Inoltre, anche se molte linee cellulari differenti sono disponibili, vi è diversità significativa tra anche un singolo tipo di cellula all&#39;interno un intero organismo. Le cellule endoteliali in particolare mostrano comportamenti diversi in base al fatto che la linea arterie o vene e che tipo di flusso è presente nel recipiente 3. Ancor più pressante da una prospettiva evolutiva, tuttavia, è che la stragrande maggioranza delle linee cellulari disponibili sono derivati ​​da tessuti adulti (vedi, ad esempio tegli collezione disponibile tramite ATCC). Queste linee cellulari adulte probabilmente non ricapitolano la natura dinamica dei loro precursori embrionali. La rapida evoluzione spazio-temporali pattern di espressione genica osservati durante lo sviluppo suggeriscono anche che una cellula endoteliale da un organo in un determinato stadio di sviluppo non può comportarsi allo stesso modo di una cellula endoteliale, da quello stesso organo, in una fase di sviluppo diverso. Come alternativa all&#39;utilizzo di linee cellulari immortalizzate disponibili in commercio, cellule primarie possono essere isolate dal tessuto specifico di interesse. Tra i vantaggi di questa tecnica, queste cellule primarie possono essere isolati da un organo specifico o anche parte di un organo in qualsiasi stadio di sviluppo specifico. Inoltre, queste cellule primarie possono essere isolati dalla grande varietà di animali transgenici disponibili, permettendo lo studio in vitro del gene knockout e knock-in, evitando altri problemi come l&#39;efficienza di trasfezione. Non sorprende che moltitecniche sono stati pubblicati in dettaglio come isolare specifici tipi di cellule 4,5. In generale, queste tecniche implicano la raccolta della regione di interesse, dissociare le cellule, tagging il tipo cellulare specifico di interesse, e isolando quelle celle per ulteriori analisi. Per studiare una popolazione precoce embrionale delle cellule endoteliali coronariche, abbiamo ridimensionato una tecnica 4 precedentemente pubblicato per l&#39;uso con un organo più piccolo. Con questa procedura in scala ridotta, possiamo isolare le cellule endoteliali coronariche dal cuore embrionale in stadi embrionali specifici. Queste cellule possono quindi essere utilizzate in saggi tradizionali endoteliali, quali le analisi di migrazione. Fino ai primi di linee cellulari embrionali sempre più frequentemente, lavorando con le cellule primarie è una tecnica di inestimabile.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Timed-pregnant mice</td>
			<td></td>
			<td></td>
			<td>To be dissected at the embryonic stage of interest</td>
		 </tr>
		 <tr>
			<td>Anti-mouse CD31</td>
			<td>BD Bioscience</td>
			<td>553370</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat IgG Dynabeads</td>
			<td>Invitrogen</td>
			<td>110-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagen</td>
			<td>BD Bioscience</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Cellgro </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich </td>
			<td>F2442 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endothelial cell growth medium</td>
			<td>(made in lab as described in notes)</td>
			<td></td>
			<td>DMEM containing 20% FBS, 5 &mu;M &beta;-mercaptoethanol, 50 &mu;g/ml ECG, and 1x penicillin/streptomycin</td>
		 </tr>
		 <tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ECGS</td>
			<td>Biomedical Technologies, Inc.</td>
			<td>BT-203</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>384-well culture plate</td>
			<td>Greiner</td>
			<td>T-3037-6</td>
			<td>Plate was prepared with a thin collagen gel, as described in 6; working surface area 10 mm2</td>
		 </tr>
		 <tr>
			<td>Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>Use at a final concentration of 1.5 mg/ml</td>
		 </tr>
		 <tr>
			<td>M199, 10x</td>
			<td>Invitrogen</td>
			<td>11825-015</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Syto-16</td>
			<td>Invitrogen </td>
			<td>S7578 </td>
			<td>Used as directed in 13 </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Stereoscopic microscope</td>
			<td>Nikon </td>
			<td>SMZ645</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell culture incubator</td>
			<td>Thermo </td>
			<td>3110 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>DynaMag</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Table-top centrifuge</td>
			<td>Thermo</td>
			<td>75002430</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation of embryonic ventricular endothelial cells

Coltura cellulare ha notevolmente migliorato la nostra capacità di valutare le singole popolazioni di cellule in condizioni di coltura miriade. Mentre le linee cellulari immortalizzate offrono vantaggi significativi per la loro facilità di utilizzo, queste linee cellulari non sono disponibili per tutti i potenziali tipi di cellule. Isolando cellule primarie di una regione di interesse, in particolare da un topo transgenico, studi più sfumate possono essere eseguite. La tecnica di base consiste dissezione dell&#39;organo o organo parziale di interesse (ad esempio il cuore o una regione specifica del cuore) e dissociare l&#39;organo di singole cellule. Queste cellule vengono poi incubate con perline magnetiche coniugate ad un anticorpo che riconosce il tipo cellulare di interesse. Le cellule di interesse possono quindi essere isolati con l&#39;uso di un magnete, con una breve incubazione tripsina dissociare le cellule dalle perline. Queste cellule isolate possono essere coltivate e analizzati come desiderato. Questa tecnica è stata originariamente progettata per adulto organo del mouses ma può essere facilmente ridotta per uso con organi embrionali, come dimostrato qui. Poiché il nostro interesse è nel sistema vascolare coronarico di sviluppo, abbiamo voluto studiare questa popolazione di cellule durante le fasi embrionali specifici. Così, il protocollo originale ha dovuto essere modificata per essere compatibile con le piccole dimensioni dei ventricoli embrionali e la bassa resa potenziale delle cellule endoteliali in queste fasi di sviluppo. Utilizzando questo approccio in scala ridotta, abbiamo valutato coronarica plesso rimodellamento ventricolare in cellule endoteliali embrionali transgeniche.

Utilizzando un anticorpo specifico per endoteliale che riconosce CD31, le cellule endoteliali sono state isolate dai ventricoli del embrionali (E) 13.5 (Figura 1A) e 18,5 (Figure 1B-1D) embrioni. Quando cresciuto su un piatto di coltura trattata, queste cellule rimangono arrotondate e formerebbero un pattern di ciottoli se vicino confluenti (Figura 1A). Diluire il collagene è stato utilizzato anche per rivestire i pozzetti, e mentre le cellule endoteliali aderirà ad e...

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

Isolation of Embryonic Ventricular Endothelial Cells

Coltura cellulare primaria è una tecnica utile per analizzare specifiche popolazioni di cellule, particolarmente da embrioni di topo transgenico a stadi specifici dello sviluppo. Qui, ventricoli embrionali vengono sezionati e dissociato, e perle di anticorpi coniugati riconoscere e separare le cellule endoteliali per ulteriori analisi.

Isolamento della embrionali ventricolari cellule endoteliali

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

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9.3K Views.  University of North Carolina at Chapel Hill. Coltura cellulare primaria è una tecnica utile per analizzare specifiche popolazioni di cellule, particolarmente da embrioni di topo transgenico a stadi specifici dello sviluppo. Qui, ventricoli embrionali vengono sezionati e dissociato, e perle di anticorpi coniugati riconoscere e separare le cellule endoteliali per ulteriori analisi.

Video: Isolation of Embryonic Ventricular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

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

Isolamento di cellule umane endoteliali della vena ombelicale (HUVEC)

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

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Biologia

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Cellule embrionali staminali endoteliali derivati ​​dalle cellule per il trattamento di ischemia degli arti posteriori

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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.

Isolamento di cellule endoteliali valvolare

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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

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

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

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

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

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

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

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

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

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolamento di cellule murine endoteliali Valve

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

Isolation 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.

Isolamento e colture primarie di cellule di topo aortica endoteliali

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

Isolation of Mouse Coronary Endothelial Cells

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

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

Isolamento di cellule di topo coronarica endoteliali

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 of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...