Questa ricerca è sostenuta dal riconoscimento alla CARRIERA NSF, la Fondazione Hartwell, e l&#39;American Heart Association (# 0830384N).

1. Preparazione <ol><li> Autoclave in uno strumento vassoio coperto i seguenti elementi: <ol><li> Forcipe del tessuto seghettato - Per la gestione del tessuto volantino </li><li> Forbici dei tessuti (8 cm) - Per tagliare i tessuti depliant e cuspidi </li><li> Tamponi di cotone - per isolare lo strato endoteliale dal volantino o cuspide </li></ol></li><li> Fai la soluzione sterile collagenasi <ol><li> Aggiungere 4,0 grammi di polvere DMEM a 250 ml di 18 MΩ acqua. </li><li> Aggiungere 1,11 grammi di bicarbonato di sodio. </li><li> Aggiungi (600 U / mL) 180.000 unità di collagenasi. </li><li> Aggiungere 1% (3 ml) Penicillina / Streptomicina. </li><li> Regolare il pH della soluzione a 7,2. </li><li> Portare la soluzione fino a 270 ml. </li><li> Sterilizzare la soluzione passa attraverso un filtro 0,2 micron. </li><li> Aggiungere 10% (30 ml) di siero fetale bovino sterile. </li></ol></li><li> Rendere sterile medio delle cellule endoteliali <ol><li> Aggiungere 6,7 grammi di polvere DMEM a 400 ml di 18 MΩ acqua. </li><li> Aggiungere 1,85 grammi di bicarbonato di sodio. </li><li> Aggiungi (50 U / mL) 25.000 unità di eparina. </li><li> Aggiungere 1% (5 ml) Penicillina / Streptomicina. </li><li> Regolare il pH della soluzione a 7,2. </li><li> Portare la soluzione fino a 450 ml. </li><li> Sterilizzare la soluzione passa attraverso un filtro 0,2 micron. </li><li> Aggiungere 10% (50 mL) di sterili siero fetale bovino. </li></ol></li><li> Rendere sterile medio delle cellule interstiziali <ol><li> Aggiungi 13,37 grammi di polvere DMEM a 800 ml di 18 MΩ acqua. </li><li> Aggiungere 3,7 grammi di bicarbonato di sodio. </li><li> Aggiungere 1% (10 ml) Penicillina / Streptomicina. </li><li> Regolare il pH della soluzione a 7,2. </li><li> Portare la soluzione fino a 900 ml. </li><li> Sterilizzare la soluzione passa attraverso un filtro 0,2 micron. </li><li> Aggiungere 10% (100 ml) di siero bovino sterile. </li></ol></li></ol> 2. Isolamento dei lembi della valvola cardiaca <ol><li> Accise radice aortica immediatamente e in modo asettico dal cuore dopo sacrificio. </li><li> Sciacquare la radice aortica di sangue freddo con DPBS sterile. E &#39;assolutamente necessario rimuovere tutti i componenti del sangue il più presto possibile per limitare la morte VEC e la contaminazione batterica. Antibiotici e antimicotici non è consigliabile in questa fase dal momento che sono potenzialmente dannose per l&#39;VEC. </li><li> Isolare i lembi della valvola (3 per ogni valvola) direttamente dalla radice e metterlo in un tubo sterile 15 ml conica riempita con 12 mL DPBS freddo. Agitare più volte per rimuovere i detriti e riempire con DPBS fresca. </li><li> Trasporto al laboratorio in ghiaccio. </li><li> Al suo arrivo in laboratorio, posto il contenitore con il tessuto sotto la cappa sterile. </li></ol> 3. Isolamento di strato endoteliale <ol><li> Riempire un piatto sterile da 35 mm con 3 ml di soluzione collagenasi freddo per valvola (3 volantini). </li><li> Mettere tutti e tre i volantini dal tubo 15 ml nel piatto riempito con la soluzione di collagenasi. </li><li> Incubare il tessuto per 5-10 minuti a 37 ° C. </li><li> Rimuovere delicatamente lo strato endoteliale ruotando un tampone asciutto e sterile sulla superficie del foglio. Il senso di rotazione e la quantità di taglio applicata è fondamentale per la purezza del campione. La rotazione del tampone dovrebbe essere in una direzione opposta al movimento lineare della mano creando un taglio controllato. Questo taglio è ciò che solleva le cellule endoteliali dal tessuto. La quantità di forza applicata dovrebbe essere sufficiente per sentire la resistenza del tessuto, ma non penetrare la membrana basale. </li><li> Occasionalmente, tamponare il tampone all&#39;interno della soluzione collagenasi per staccare le cellule dalle fibre punta. Dopo tampone è completo, la trama dello strato endoteliale dovrebbe sentirsi un po &#39;più agevole rispetto a prima. </li><li> Raccogliere la sospensione cellulare / collagenasi e trasferirli in un nuovo tubo sterile 15ml. </li><li> Centrifugare le provette a 1000 giri per 5 minuti per far sedimentare le cellule isolate e aspirare il surnatante. Se isolare cellule interstiziali e, eseguire tale protocollo, mentre queste cellule vengono centrifugate. </li><li> Aggiungere 3 ml di medium suina endoteliali ai 15 mL, centrifugare una seconda volta, e media aspirare. Questo aiuta a centrifugazione secondo filtro parte del materiale non desiderato come le fibre punta. </li><li> Risospendere le cellule endoteliali centrifugati in 5 ml di terreno suina endoteliali e le cellule piastra in un pre-rivestito T-25 pallone con collagene (utilizzare 1 fiasco per provetta). </li><li> Lasciate che le cellule crescono, almeno 2-3 giorni prima di cambiare il medium endoteliale. Questo aiuta le cellule recuperare e dividere in quanto il processo di isolamento è abbastanza dura e la resa delle cellule può essere bassa. E &#39;fondamentale per le cellule passaggio vicino confluenza quanto l&#39;inibizione contatto potrebbe portare alla trasformazione cellulare. </li></ol> 4. Preparazione di 60 Dish Cultura mm per isolamento laterale specifico <ol><li> La linea 60 millimetri di vetro piastre di Petri con un foglio di alluminio (2 piatti di vetro per leaflet). Il foglio di alluminio aiuta a rimuovere la paraffina, in modo che il piatto di vetro di Petri possono essere riutilizzati per altri isolamenti. </li><li> Luogo perline paraffina all&#39;interno dei piatti, circa mezzo pieno, e la copertura per sterilizzazione in autoclave. </li><li> Una volta che l&#39;autoclave è completa e paraffina viene fuso, spostare il piatto insieme ad una superficie piana fredda. Come la paraffina si raffredda, si indurisce e creare uno strato che sosterrà punture d&#39;ago. </li><li> Dopo 30 minuti, il piatto sterilizzato può quindi essere utilizzato come una camera di isolamento per immobilizzare il tessuto foglio. </li></ol> 5. Isolamento di Side strato endoteliale specifico <ol><li> Rimuovere i volantini dai tubi 15ml e posto su piatto preparato cultura 60mm per lato isolamento specifico. </li><li> Manipolare il foglio in modo che il lato ventricularis è a faccia in giù sulla superficie paraffina lasciando esposta la parte fibrosa. Pin i bordi del foglio per esporre lo strato endoteliale. </li><li> Mettere qualche goccia di collagenasi fredda su ogni (rivolto verso l&#39;alto) della superficie endoteliale e incubare il tessuto per 5-10 minuti a 37 ° C. </li><li> Come prima, rimuovere delicatamente lo strato endoteliale ruotando un tampone asciutto e sterile sulla superficie del foglio. Il senso di rotazione e la quantità di taglio applicata è fondamentale per la purezza del campione. La rotazione del tampone dovrebbe essere in una direzione opposta al movimento lineare della mano creando un taglio controllato. Questo taglio è ciò che solleva le cellule endoteliali dal tessuto e nient&#39;altro. La quantità di forza applicata dovrebbe essere sufficiente per sentire la resistenza del tessuto, ma non penetra all&#39;interno del tessuto. </li><li> Occasionalmente, tamponare il tampone all&#39;interno della soluzione collagenasi per staccare le cellule dalle fibre punta. Dopo tampone è completo, la trama dello strato endoteliale dovrebbe sentirsi un po &#39;più agevole rispetto a prima. </li><li> Raccogliere la sospensione cellulare / collagenasi e trasferirli in un nuovo tubo sterile 15ml. Indicare specificità lato sull&#39;etichetta (volantini dalla valvola stessa può essere raggruppate). </li><li> Trasferire i volantini per un piatto di nuova cultura in modo che il lato ventricularis è ora esposta e ripetere i passaggi (5,3-5,6). </li><li> Una volta che tutti sospensione cellulare / collagenasi viene raccolto, centrifugare le provette a 1000 rpm per 5 minuti per far sedimentare le cellule isolate e aspirare il surnatante. Se isolare cellule interstiziali e, eseguire tale protocollo, mentre queste cellule vengono centrifugate. </li><li> Aggiungere 3 ml di medium suina endoteliali ai tubi, centrifugare una seconda volta, ed aspirare media. Questo aiuta a centrifugazione secondo filtro parte del materiale non desiderato come le fibre punta. </li><li> Risospendere le cellule endoteliali centrifugati in 5 ml di terreno suina endoteliali e le cellule piastra in un pre-rivestito T-25 pallone con collage (utilizzare 1 fiasco per provetta). </li><li> Lasciate che le cellule crescono, almeno 2-3 giorni prima di cambiare il medium endoteliale. Questo aiuta le cellule recuperare e dividere in quanto il processo di isolamento è abbastanza dura. Se notate la resa sia molto bassa, in considerazione di passare a un fiasco più piccolo o pozzetti in quanto cellula-cellula adesione promuove la crescita cellulare. Ricordatevi di passaggio vicino confluenza quanto l&#39;inibizione contatto potrebbe portare alla trasformazione cellulare. </li></ol> 6. Isolamento di cellule interstiziali <ol><li> Riempire una provetta sterile 15 ml con 10 ml di soluzione di collagenasi per valvola (3 volantini). </li><li> Dopo strofinare i volantini delle cellule endoteliali, immediatamente posto nel tubo appropriato 15ml con la soluzione di collagenasi. </li><li> Incubare per circa 12 a 18 ore (agitare gentilmente se desiderato). </li><li> Mescolare il tessuto degradato delicatamente con una pipetta sierologica fino sospensione cellulare / collagenasi diventa omogeneizzato. Questa omogeneizzazione aiuta a rompere il tessuto e rilasciare le cellule interstiziali. </li><li> Centrifugare il tessuto digerito per 5 minuti a 1000 rpm e aspirare il surnatante. </li><li> Aggiungere 5 mL medio suina interstiziale ai tubi 15 ml, centrifugare una seconda volta, ed aspirare supernate. </li><li> Risospendere le cellule endoteliali centrifugati in 5 ml di terreno suina interstiziale e la piastra le celle di una T-75 pallone (utilizzare 1 fiasco per provetta). </li><li> Lasciate che le cellule crescono, almeno 1-2 giorni prima di cambiare il medium interstiziali delle cellule. Ci sarà detriti tessuto molto di più che con le cellule endoteliali, ma, che ci si aspetta. Le cellule dovrebbe aumentare anche la confluenza di più velocemente le cellule endoteliali a causa del rendimento delle cellule e la loro natura. </li></ol> 7. Immagini rappresentante <img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Figura 1" /> Figura 1. La morfologia delle cellule isolate a 2-3 giorni dopo l&#39;isolamento. (A) VEC mostrano una morfologia tipica endoteliale e formare gruppi per promuovere la crescita. (B) VIC morfologia è simile a miofibroblasti che sono generalmente a forma di fuso e diffusa in modo uniforme in tutto il pallone. <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Figura 2" /> Figura 2. La morfologia delle cellule isolate a confluenza. (A) VEC mostrano una morfologia tipica endoteliali che sono generalmente di ciottoli e il contatto crescita inibita. (B) VIC morfologia è simile a miofibroblasti che sono generalmente a forma di fuso e di non contattare la crescita inibita. <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Figura 3" /> Figura 3. Le caratteristiche di funzione delle cellule isolate 6. (A) VEC sono associati con alti livelli di assorbimento acetilato LDL (rosso). (B) VIC sono associate a bassi livelli di LDL acetilato assorbimento. <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Figura 4" /> Figura 4. Marcatori cellulari di cellule isolate 6. (A) fenotipo VEC è contribuito alla colorazione positiva per il fattore di von Willebrand (verde), blu (nuclei). (B) fenotipo VIC è contribuito a colorazione negativa per il fattore di Von Willebrand. <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Figura 5" /> Figura 5. Marcatori cellulari di cellule isolate. (A) fenotipo VEC è contribuito a colorazione negativa per alpha SMA (verde), blu (nuclei). (B) fenotipo VIC è contribuito alla colorazione positiva per la SMA alfa. <table border="1"><tbody><tr><td> Dissociazione agente </td><td> Dissociazione Tecnica </td><td> Di raccolta delle cellule </td><td> Quantità di cellule </td><td> Cellula Purezza </td><td> Contaminazione </td></tr><tr><td> EDTA (3 mm) senza CaCl 2 </td><td> 5, 20, 60 min. Inoltre prima di CaCl 2 </td><td> 20, 60, 120 min. prima della raccolta </td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td> EDTA (6 mm) senza CaCl 2 </td><td> 5, 20, 60 min. Inoltre prima di CaCl 2 </td><td> 20, 60, 120 min. prima della raccolta </td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td> Tripsina-EDTA (0,5 g / L) </td><td> 5, 10, 15 min. prima di disattivazione </td><td> Media raccolti immediatamente </td><td> + </td><td> + </td><td> + + </td></tr><tr><td> Collagenasi II (300 U / mL) </td><td> 5, 10, 15 min. prima di disattivazione </td><td> Media raccolti immediatamente </td><td> -, +, + + </td><td> -, + +, + </td><td> + </td></tr><tr><td> Collagenasi II (600 U / mL) </td><td> 5, 10, 15 min. prima di disattivazione </td><td> Media raccolti immediatamente </td><td> +, + +, + + + </td><td> + +, + +, - </td><td> - </td></tr></tbody></table> Tabella 1. Risultati preliminari di protocolli delle cellule endoteliali valvolare isolamento.

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Nessun conflitto di interessi dichiarati.

La comprensione della biologia valvolare è stata compromessa da difficoltà tecniche di isolamento e la coltura popolazioni di puro valvolare cellule endoteliali. Tecniche di isolamento tipico coinvolgere digestione enzimatica della matrice sottostante basale o chimica di dissociazione dei legami adesivi endoteliale 2,3. Isolamento esperimenti preliminari sono stati valutati qualitativamente variando agenti dissociazione e periodi di incubazione. I risultati di questi esperimenti hanno dimostrato che l&#39;E...

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<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
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<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
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<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
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<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
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<td>DPBS</td>
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<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
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<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
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<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
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<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
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<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
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<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
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<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
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<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
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<td>VWR</td>
<td>89000-310</td>
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<td>Paraffin Embedding Wax</td>
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<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
<td>&nbsp;</td>
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<td>Precision Glide Needles</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>305165</td>
<td>&nbsp;</td>
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<td>500 mL Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-985</td>
<td>&nbsp;</td>
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<td>1L Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-986</td>
<td>&nbsp;</td>
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<td>Tissue Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11023-15</td>
<td>&nbsp;</td>
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<td>FSC Tweezers #5</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-00</td>
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isolation of valvular endothelial cells

Valvole cardiache è l&#39;unico responsabile per il mantenimento del flusso unidirezionale del sangue attraverso il sistema cardiovascolare. Queste sottili, tessuti fibrosi sono sottoposti a notevoli sollecitazioni meccaniche come aprire e chiudere diversi miliardi di volte in un ciclo di vita. L&#39;incredibile resistenza di questi tessuti è dovuta alla valvolare residente endoteliale (VEC) e le cellule interstiziali (VIC) che costantemente la riparazione e rimodellare in risposta a segnali meccanici locali e biologici. Solo di recente abbiamo cominciato a capire i comportamenti unica di queste cellule, per il quale nella sperimentazione in vitro ha svolto un ruolo chiave. Particolarmente impegnativo è l&#39;isolamento e la coltura di VEC. Particolare cura deve essere utilizzato dal momento in cui il tessuto viene rimosso dal host tramite placcatura finale. Qui vi presentiamo i protocolli per l&#39;isolamento diretto, isolamento laterale specifico, la cultura, e la verifica delle popolazioni pura VEC. Noi usiamo digestione enzimatica seguita da una tecnica delicata raschiatura tampone per staccare le cellule di superficie. Queste cellule vengono poi raccolti in un tubo e centrifugati in un pellet. Il pellet viene quindi risospeso e placcato in fiasche cultura pre-rivestito con collagene I matrice. Fenotipo VEC è confermata dal contatto inibito la crescita e l&#39;espressione di marcatori endoteliali specifici come PECAM1 (CD31), fattore di von Willebrand (vWF), e l&#39;espressione negativa di alfa-actina del muscolo liscio (α-SMA). Le caratteristiche funzionali di VEC sono associati con alti livelli di LDL acetilato. A differenza di cellule endoteliali vascolari, VEC hanno la capacità unica di trasformare in mesenchima, che normalmente si verifica durante la formazione embrionale valvola 1. Questo può verificarsi anche durante confluenti in modo significativo dopo prolungata coltura in vitro, per cui la cura dovrebbe essere fatto per il passaggio o in prossimità confluenza. Dopo l&#39;isolamento VEC, popolazioni pure di VIC può quindi essere facilmente acquisita.

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

Isolation of Valvular Endothelial Cells

Forniamo un metodo per isolare e popolazioni coltura pura di cellule endoteliali valvola cardiaca (VEC). VEC può essere isolato da entrambi i lati della cuspide o depliant e subito dopo, alla base di cellule interstiziali (VIC) isolamento è semplice.

Isolamento di cellule endoteliali valvolare

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

Research

JoVE Journal

Biology

20.2K Views.  Cornell University. Forniamo un metodo per isolare e popolazioni coltura pura di cellule endoteliali valvola cardiaca (VEC). VEC può essere isolato da entrambi i lati della cuspide o depliant e subito dopo, alla base di cellule interstiziali (VIC) isolamento è semplice.

Video: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

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

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 ...

Ricerca

Biologia

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

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

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

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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

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

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 Embryonic Ventricular Endothelial Cells

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

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

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 ...

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 ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Isolamento simultaneo di cardiomiociti di alta qualità, cellule endoteliali e fibroblasti da un cuore di ratto adulto

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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

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

Isolamento e cultura delle cellule endoteliali aortiche primarie dai suini in miniatura

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