Name: isolation and culture of primary endothelial cells from canine arteries and veins
Uploaded: 2016-11-18T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

The authors would like to acknowledge Hans de Graaf and Tomas Veenendaal for their technical assistance in culturing the ECs.

Ethik-Erklärung: Die Blutgefäße in dieser Studie verwendet wurden, als überschüssige Material geerntet wurden aus frischen Hunde-Kadaver (n = 4) von gesunden Hunden für andere, unabhängige Forschung (Universität 3R Politik) euthanasiert. Aberrant Blutgefäße (intra- und extrahepatischen portosystemischen Shunts, n = je 1) vorgestellt wurden für Haustiere der Universität Utrecht an der Universitätsklinik von Hunden post-mortem nach informierte Zustimmung der Eigentümer geerntet. 1. Isolierung und Kultur von primären Canine Endothelial Cells <ol><li> Precoat-6-well - Platten mit 2 ml / Vertiefung 0,5% w / v Gelatine und mit einer angefeuchteten Atmosphäre von 5% CO 2 bei 37 ° C für 2 h in einem Brutschrank belassen. Entfernen Lösung überschüssige Gelatine vor der primären Zellen Aussaat. </li><li> Aseptisch entfernen Sie das Blutgefäß (e) von Interesse (zB Aorta, Hohlvene, Vena porta) von einem frischen Eckzahn kadaver (Abbildung 1A). Aufrechterhaltung der Anatomie des Gefäßsystemsindem gerade Zange an beiden Enden des Schiffes von Interesse. Unnötige Manipulation des Gewebes mit einer Pinzette Schädigung der Endothelzellen zu verhindern. <ol><li> Schneiden Sie das geklemmt Gefäß an beiden Enden mit chirurgischen Scheren und vom kadaver entfernen. Transport in Hanks Balanced Salt Solution (HBSS) auf Eis. HINWEIS: Eine Schiffslänge von ca. 5 cm Für dieses Verfahren wird bevorzugt, aber diejenigen, die 1 cm messen auch genügend Zellen zur Kultur bieten. </li></ol></li><li> Übertragen Sie das Blutgefäß in eine Petrischale gefüllt mit eiskaltem HBSS. Mit chirurgische Scheren, entfernen Sie alle anhaftendem Gewebe und Fett von der Außenseite des Gefäßes, halten das Gefäß selbst intakt (1B). Stellen Sie sicher, dass das Schiff jederzeit deutlich sichtbar ist beim Schneiden: beiseite ziehen das umgebende Gewebe mit einer Klammer oder einer Zange für optimale Sicht. <ol><li> Schließen Sie alle Zweige des Schiffes mit Ligatur (zB Polyglactin 3-0) und sie anschließend zu entfernenmit chirurgischen Schere oder einem Skalpell. </li></ol></li><li> Ein Gefäßende mit einem gekrümmten Halsted Moskito Zange, klemmen die Spitze der Zange an dem anderen Ende des Behälters sorgfältig eingeben und dann zurückziehen, langsam das Gefäß Invertieren bis er vollständig von innen nach außen (1C-G) ist. Die Außenseite besteht nun aus der endothelialen Zellschicht. Wenn sich eine Schwierigkeit beim Umdrehen des Behälters angetroffen wird, wieder in HBSS versenken Reibung zu reduzieren. </li><li> Platzieren Sie Tabaksbeutelnähte an beiden Enden des Schiffes es vollständig zu schließen und die Exposition von Nicht-endothelialen Gefäßgewebe zu dem Aufschlussverfahren (Abbildung 1H) zu verhindern. Verwenden Sie die Ligatur Enden des invertierten Gefäß zu manipulieren. </li><li> Wenn die Verdauung und Kultur später durchgeführt wird, das invertierte Kryokonservierung Gefäß vor dem Verdau Protokoll (Schritt 1.7). <ol><li> Legen Sie das invertierte Schiff in einem Kryoröhrchen und füllen mit Zellkultur Einfriermedium. Einfrieren bis -80 ° C, um ein Einfrieren Co mitntainer. Bei -80 ° C, wenn Schiffe sind innerhalb einer Woche verwendet werden. Für die langfristige Lagerung Ort Kryovials bei -180 ° C. Wenn die EG-Isolierung durchgeführt wird, tauen die Kryovials schnell in einem Wasserbad (37 ° C) und sofort in eiskaltem HBSS platzieren. Gehen Sie wie in 1.7 angegeben. Hinweis: Beachten Sie, dass die Gesamtausbeute von ECs nach der Isolierung geringer sein wird durch den Verlust der Lebensfähigkeit während des Gefrierprozesses. </li></ol></li><li> Übertragen Sie das Gefäß in ein 50 - ml - Röhrchen und spülen Sie es zweimal in HBSS Erythrozyten (oder Rest Einfriermedium bei Auftauen) (1I) zu entfernen. </li><li> Digest das Gefäß in einer 50-ml-Röhrchen mit einer Lösung von 30 ml Kollagenase Typ II (0,15 U / ml) und Dispase (0,15 U / ml) in Canine Endothelial Cells Growth Medium (CECGM) für 1 h bei 37 ° C mit intermittierender sanften Agitation. </li><li> Entfernen des Behälters aus dem Rohr und zentrifugieren die Zellsuspension für 5 min bei 250 x g. </li><li> Zellpellet in CECGM einnd Samen 2 ml Zellsuspension pro Well (1 - 3 Brunnen je nach Gefäßgröße). Kultur der Zellen bei 37 ° C in einer befeuchteten Atmosphäre mit 5% CO 2 in Luft und das Kulturmedium zweimal pro Woche ändern. </li><li> Passage der Zellen, wenn eine Konfluenz von 70-80% erreicht ist. <ol><li> Waschen Sie die Zellen mit vorgewärmtem HBSS tot oder nicht-anhaftenden Zellen zu entfernen. In 200 ul rekombinanter Zell-Dissoziation Enzym (1x) in jede Vertiefung und legen zurück in den Inkubator für 5 Minuten oder bis alle Zellen abgelöst werden. </li><li> Übertragen Sie die Zellen in ein 15-ml-Tube und stoppen Trypsinierung durch Zugabe von 10 ml Kulturmedien (CEGM) mit 10% fötalem Kälberserum (FCS). Zentrifuge für 5 Minuten bei 250 x g. Weiter wird die Kultur im Gelatine vorbeschichtet Platte oder Kolben. </li></ol></li></ol> 2. Charakterisierung <ol><li> Kultur Canine Aortic Endothelial Cells (CnAOECs) und die Morphologie der primären und kommerziellen Zelllinien zweimal wöchentlich mit einem Mikroskop vergleichen. HINWEIS: Ter wächst typisches Muster von ECs in Patches lassen sich am besten beobachtet werden, wenn eine Konfluenz von&gt; 70% erreicht ist. <ol><li> Kultur CnAOECs in CECGM auf einem 0,5% w / v Gelatine vorbeschichtet T75-Kolben. Passage-Zellen einmal in der Woche, wenn die Konfluenz 70 - 80%. <ol><li> Für Passagierung, waschen Sie die Zellen einmal mit vorgewärmter HBSS und 1 ml rekombinanten Zell-Dissoziation Enzym (1x). Der Kolben wird wieder in den Inkubator für 5 Minuten oder bis alle Zellen abgelöst werden. Inactivate Trypsin von 10 ml Kulturmedium Zugabe (CEGM) mit 10% FCS. Zentrifugieren Sie die Zellsuspension für 5 Minuten bei 250 · g, den Überstand verwerfen und Resuspendieren der Zellen in 1 ml Kulturmedium. </li><li> Nehmen Sie ein 10-ul-Aliquot der Zellsuspension und verdünnt 1: 1 mit 0,4% Trypanblau. Zählen Sie die Zellen eines automatisierten Zellzähler verwenden, und die Platte aus 4,0 x 10 5 lebensfähigen Zellen in einem neuen T75 - Flasche. 10 ml vorgewärmtes CECGM in den Kolben und die Kultur bei 37 ° C in einer befeuchteten Atmosphäre mit 5% CO 2. </li></ol></li></ol></li><li> Isolieren von RNA aus Durchgang 1 der kultivierten CaPECs und CnAOECs. <ol><li> Sammeln Sie mindestens 1 x 10 3 Zellen als Pellet, fügen Sie 20 ul der Probenvorbereitung Reagenz (SPR) und Inkubation für 1 min die Zellen zu lysieren. Nach der Inkubation sorgfältig sammeln Zelllysat Gesamt-RNA und lagern bei -70 ° C enthält. </li></ol></li><li> Konvertieren von mRNA in cDNA eine cDNA - Synthese - Kit (zB iScript) nach den Anweisungen des Herstellers. Shop cDNA bei 4 ° C bis zu einer Woche oder bei -20 ° C für langfristige, bis bereit, die qPCR auszuführen. </li><li> Messen Sie die Genexpression der endothelialen Marker CD31 Endothel-Zell-Ursprung zu bestätigen. Führen Versuchsaufbau und die Quantifizierung mit Hilfe der MIQE précis Richtlinien 10. Zur Normalisierung, messen die Expression von Referenzgenen GAPDH, RPS19 und B2MG 11. <ol><li> Bereiten Sie eine 10-fache Verdünnung der erhaltenen cDNA in nukleasefreiem Wasser. </li><li> Bereiteneine 4-fache Verdünnung von gepoolten cDNA-Proben für die Standard-Linie und nukleasefreiem Wasser als Nicht-Template-Steuerung verwenden. Verdünnen Sie die Proben fünfmal einen 50-fachen Gesamtverdünnung für qPCR Reaktionen zu erreichen. Pipette 10 ul - Reaktionen in doppelter Ausfertigung in einer 384-Well - Format unter Verwendung von 4 &amp; mgr; l cDNA und 6 &amp; mgr; l des Fluorophors mit 20 pmol reverser gemischt und Vorwärts - Primer (Tabelle 1). </li><li> Stellen Sie das Programm auf 95 ° C für 5 min für Taq-Polymerase-Aktivierung, gefolgt von 39 Zyklen von 10 sec bei 95 ° C für die Denaturierung und 30 sec bei Tm zum Glühen und Dehnung. Die Tm für jeden Primersatz ist in Tabelle 1 gezeigt. </li><li> Perform Kurve Schmelzen analysiert jede Lauf folgende nur ein Produkt, um sicherzustellen, wird verstärkt. Normalisieren der Expressionsniveaus der Proben die durchschnittliche relative Menge der Referenzgene verwenden, und berechnen die ACt wenn Reaktionswirkungsgrad zwischen 95% und 105% liegt. </li></ol></li><li> Beurteilen Sie EC-Funktionalität mit einem angiogenesis Test. <ol><li> Zugabe von 10 &amp; mgr; l der extrazellulären Matrix zu jeder Vertiefung einer vorgekühlten Angiogenese Dia- und verteilt mit einer Pipettenspitze die Oberfläche der Vertiefung zu bedecken. Halten Sie die extrazelluläre Matrix auf Eis und vermeiden, dass die Einführung von Luftblasen in das Gel. In einem Inkubator bei 37 ° C mit 5% CO 2 für 30 min erstarren das Gel durch den Schieber in eine Petrischale mit Wasser getränkten Papiertüchern platzieren. </li><li> In 1,0 x 10 4 primäre ECs in 50 ul Endothelial Growth Medium pro Vertiefung. Inkubieren der Objektträger 6 Stunden bei 37 ° C mit 5% CO 2. </li><li> Nach 6 Stunden Fotos mit einer 20-facher Vergrößerung nehmen, um sicherzustellen, das Ganze gut in das Bild aufzunehmen. </li></ol></li></ol>

<ol>
	<li>Haidara, M. A., Assiri, A. S., Yassin, H. Z., Ammar, H. I., Obradovic, M. M., Isenovic, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Failure+Models:+Traditional+and+Novel+Therapy.">Heart Failure Models: Traditional and Novel Therapy.</a> Curr. Vasc. Pharmacol. 13 (5), 658-669 (2015).</li><li>van Steenbeek, F. G., van den Bossche, L., Leegwater, P. A., Rothuizen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+liver+shunts+in+dogs+elucidate+pathways+regulating+embryonic+development+and+clinical+disorders+of+the+portal+vein.">Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein.</a> Mamm. Genome. 23 (1-2), 76-84 (2012).</li><li>Murai, A., Asa, S. A., Kodama, A., Hirata, A., Yanai, T., Sakai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+phosphorylation+of+the+mTORC2/Akt/4E-BP1+pathway+in+newly+derived+canine+hemangiosarcoma+cell+lines.">Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines.</a> BMC Vet. Res. 8 (1), 128 (2012).</li><li>Boilson, B. 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=Regulation+of+circulating+progenitor+cells+in+left+ventricular+dysfunction.">Regulation of circulating progenitor cells in left ventricular dysfunction.</a> Circ. Heart Fail. 3 (5), 635-642 (2010).</li><li>Gonzalez-Miguel, J., Morchon, R., Siles-Lucas, M., Simon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolysis+and+proliferative+endarteritis:+two+related+processes+in+chronic+infections?+The+model+of+the+blood-borne+pathogen+Dirofilaria+immitis.">Fibrinolysis and proliferative endarteritis: two related processes in chronic infections? The model of the blood-borne pathogen Dirofilaria immitis.</a> PLoS One. 10 (4), e0124445 (2015).</li><li>Sacks, T., Moldow, C. F., Craddock, P. R., Bowers, T. K., Jacob, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+radicals+mediate+endothelial+cell+damage+by+complement-stimulated+granulocytes.+An+in+vitro+model+of+immune+vascular+damage.">Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. An in vitro model of immune vascular damage.</a> J. Clin. Invest. 61 (5), 1161-1167 (1978).</li><li>Aranguren, X. 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=Unraveling+a+novel+transcription+factor+code+determining+the+human+arterial-specific+endothelial+cell+signature.">Unraveling a novel transcription factor code determining the human arterial-specific endothelial cell signature.</a> Blood. 122 (24), 3982-3992 (2013).</li><li>van Balkom, B. 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+cells+require+miR-214+to+secrete+exosomes+that+suppress+senescence+and+induce+angiogenesis+in+human+and+mouse+endothelial+cells.">Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.</a> Blood. 121 (19), 3997-4006 (2013).</li><li>Crampton, S. P., Davis, J., Hughes, C. C. <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+human+umbilical+vein+endothelial+cells+(HUVEC).">Isolation of human umbilical vein endothelial cells (HUVEC).</a> J. Vis. Exp. (3), e183 (2007).</li><li>Bustin, S. 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=MIQE+precis:+Practical+implementation+of+minimum+standard+guidelines+for+fluorescence-based+quantitative+real-time+PCR+experiments.">MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.</a> BMC Mol. Biol. 11, 74 (2010).</li><li>Brinkhof, B., Spee, B., Rothuizen, J., Penning, L. 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+and+evaluation+of+canine+reference+genes+for+accurate+quantification+of+gene+expression.">Development and evaluation of canine reference genes for accurate quantification of gene expression.</a> Anal. Biochem. 356 (1), 36-43 (2006).</li><li>Heishima, 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=MicroRNA-214+and+MicroRNA-126+Are+Potential+Biomarkers+for+Malignant+Endothelial+Proliferative+Diseases.">MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases.</a> Int. J. Mol. Sci. 16 (10), 25377-25391 (2015).</li><li>Liu, M. M., Flanagan, T. C., Lu, C. C., French, A. T., Argyle, D. J., Corcoran, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+characterisation+of+canine+mitral+valve+interstitial+and+endothelial+cells.">Culture and characterisation of canine mitral valve interstitial and endothelial cells.</a> Vet. J. 204 (1), 32-39 (2015).</li><li>van den Bossche, L., van Steenbeek, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+congenital+portosystemic+shunts:+disconnections+dissected.">Canine congenital portosystemic shunts: disconnections dissected.</a> The Veterinary Journal. 211, 14-20 (2015).</li><li>Sobczynska-Rak, A., Polkowska, I., Silmanowicz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+Vascular+Endothelial+Growth+Factor+(VEGF)+levels+in+the+blood+serum+of+dogs+with+malignant+neoplasms+of+the+oral+cavity.">Elevated Vascular Endothelial Growth Factor (VEGF) levels in the blood serum of dogs with malignant neoplasms of the oral cavity.</a> Acta Vet. Hung. 62 (3), 362-371 (2014).</li><li>Zhang, Q., 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=In+vitro+and+in+vivo+study+of+hydralazine,+a+potential+anti-angiogenic+agent.">In vitro and in vivo study of hydralazine, a potential anti-angiogenic agent.</a> Eur. J. Pharmacol. 779, 138-146 (2016).</li></ol>

The authors have no competing financial interests.

In Studien über Hunde-ECs Fokussierung der CnAOEC primäre Linie , die die endotheliale Abstammungslinien des Hundes 3, 12, Modell 13. In Studien am Menschen wird die HUVEC Kultur immer noch der Goldstandard. Offensichtlich aus Nabelschnur abgeleitet Fokussierung nur auf ECs ist eine feste Beschränkung in der kardiovaskulären Forschung. Endothelial-Zellen haben einen spezifischen Genexpressionsmuster arteriovenöse Spezifikation zu bestimmen. Um diese Unterschiede in der postnatalen Schiffe zu b...

Hunde sind als Großtiermodell für kardiovaskuläre Erkrankungen Forschung und auch von angeborener (genetischen) Gefäßanomalien 1, 2 leiden können. Um diese Krankheiten kommerziellen Endothelzelllinien studieren häufig verwendet werden , um Endothelzellen (EC) Funktionalität beurteilen. Für Hunde gibt es eine kommerzielle Endothelzelllinie verfügbar (CnAOEC), von Eckzahn Aorta abgeleitet. Diese Zelllinie ist vor allem in Studien als Kontrolle normalen ECs 3-5 verwendet. In der menschlichen kardiovaskulären Forschung sind die am häufigsten verwendeten Endothelzelllinien menschlichen Nabelschnurvenen-Endothelzellen (HUVECs) und menschlichen Nabelschnurarterie Endothelial Cells (HUAECs) aus menschlicher Nabelschnur Vene und Arterie abgeleitet sind. HUVECs wurden als Goldstandard in der Gefäßforschung seit den 1980er Jahren 6 verwendet. Sie gelten als das klassische Modellsystem zum Studium der Endothelfunktion und Krankheit Anpassung sein. Endothelial-Zellen isoliert aus verschiedenen Blutgefäße variieren in appearance und Funktionalität durch genetische Hintergrund und der Exposition gegenüber der Mikroumgebung 7. Zusätzlich werden HUVECs und HUAECs aus Nabelschnur abgeleitet sind, eine Entwicklungsgefäßstruktur, die an die Bedingungen in Bezug erwachsenen Blutgefäße möglicherweise nicht vollständig mimic sie und die Reaktion auf Krankheit ausgesetzt sind. Daher ist unzureichend in HUVECs und HUAECs zu kardiovaskulären Erkrankungen im Allgemeinen gefunden Ergebnisse zu übersetzen. Wenn die Anpassung und das Verhalten von Erwachsenen ECs studieren, primären ECs aus dem Gefäß von Interesse sollte als direkter Ansatz verwendet werden. Zur Isolierung haben diese Zellen zeigen verschiedene Methoden berichtet. Eine weit beschriebene Verfahren, die auch für HUVECs verwendet wird, wird das Spülen des Behälters mit einem enzymatischen Verdauungslösung 8. Dies führt oft zu einer Kontamination mit nicht-ECs wie glatte Muskelzellen und Fibroblasten 9. Ein anderes häufig verwendetes Verfahren zur Isolierung ist die enzymatische Verdauung von gehackten Gefäßgewebe, gefolgt von Fluoreszenz-aktivierte Zelle (FACS) , basierend auf endothelialen Zellmarker Cluster of Differentiation (CD) 31 7, 8. FACS - Sortierung und anschließender Zellkultur Sortieranlage erfordert relativ große Mengen an Zellen und ist daher nicht geeignet für die Isolierung von Endothel von kleinen Blutgefäßen. Wir haben daher ein neues robustes Verfahren zu entwickeln, zur Isolierung eines reinen endothelialen Zellpopulation aus verschiedenen Hunde- Blutgefäße mit hoher Reinheit gerichtet. Um die Effizienz des neuen Isolationsmethode testen, haben wir isoliert und rein erhalten Canine Primary Endothelzell (CAPEC) Kulturen aus verschiedenen Hunde-Arterien und Venen, große und kleine. Dieses Verfahren ermöglicht auch die Kultur von Endothelzellen aus erkrankten Ursprung und / oder abweichende Gefäße wie angeborene intra- oder extrahepatischen portosystemischen Shunts, eine häufige Erkrankung bei Hunden 2. Das Verfahren erlaubt die Isolierung von zusätzlichen relevanten Zelltypen des gleichen Gefäß wie vaskulären glatten Muskelzellen, da die meisten des Schiffes int bleibtWährend des Verfahrens handeln.

<table><tbody><tr><td>Collagenase type II</td><td>Life Technologies</td><td>17101-015</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Life Technologies</td><td>31966-021</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-050</td><td></td></tr><tr><td>Canine Endothelial Cells Growth Medium&amp;nbsp;</td><td>Cell Applications</td><td>Cn211-500</td><td></td></tr><tr><td>CnAOECs</td><td>Cell Applications</td><td>Cn304-05</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)&amp;nbsp;</td><td>GE Healthcare</td><td>16000-044</td><td></td></tr><tr><td>TrypLE Express</td><td>Life Technologies</td><td>12604-013</td><td></td></tr><tr><td>SPR</td><td>Bio-Rad</td><td>170-8898</td><td></td></tr><tr><td>iScript synthesis kit</td><td>Bio-Rad</td><td>170-8891</td><td></td></tr><tr><td>SYBR green super mix</td><td>Bio-Rad</td><td>170-8886</td><td></td></tr><tr><td>Recovery Cell Freezing Medium</td><td>Gibco/Life Technologies</td><td>12648-010</td><td>Keep on ice prior to use</td></tr><tr><td>Freezing container, Nalgene Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Surgical scissors (Mayo or Metzenbaum)</td><td>B. Braun Medical</td><td>BC555R</td><td></td></tr><tr><td>Mosquito forceps&amp;nbsp;</td><td>B. Braun Medical</td><td>FB440R</td><td></td></tr><tr><td>Mosquito forceps curved</td><td>B. Braun Medical</td><td>FB441R</td><td></td></tr><tr><td>polyglactin 3-0</td><td>Ethicon</td><td>VCP311H</td><td></td></tr><tr><td>Trypan blue</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Automated counting chamber</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Counting Slides, Dual Chamber</td><td>Bio-Rad</td><td>145-0011</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>BD356231</td><td>Slowly thaw on ice</td></tr><tr><td>&amp;micro;-Slide Angiogenesis</td><td>Ibidi</td><td>81501</td><td></td></tr><tr><td>Endothelial Growth Medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EGM-2 SingleQuot Kit&amp;nbsp;</td><td>Lonza</td><td>CC-4176</td><td></td></tr></tbody></table>

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.

Verschiedene Blutgefäße wurden erfolgreich auf das beschriebene Isolierungsprotokoll (Figur 2) unterzogen. Es war möglich, zu sezieren und Aorta, Hohlvene, Vena porta invertieren und Koronararterie von gesunden Hunden (alle Gefäße von jedem Hund, n = 4). Mit dem gleichen Ansatz ECs wurden aus zwei angeborenen portosystemischen Shunts (extrahepatische und intrahepatische, n = je 1) isoliert. Obwohl Aorta leicht invertiert wurde, waren thorakalen Aorta Segmente eine g...

Watch this Scientific Journal Video about Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins at JoVE.com

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

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.

Isolierung und Kultur von primären Endothelzellen von Canine Arterien und der

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

isolation-culture-primary-endothelial-cells-from-canine-arteries

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.2K Aufrufe.  Utrecht University. 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.

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

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.

Isolierung von Valvular Endothelzellen

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

Forschung

Biologie

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.

Isolierung und Kultivierung von pulmonaler Endothelzellen aus neugeborenen Mäusen

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 Microvascular Endothelial Tubes from Mouse Resistance Arteries

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

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

Microvascular Endothelial Isolierung von Rohren aus Maus Widerstand Arterien

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

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolierung von primären Lungenendothelzellen der Maus

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunologie und Infektion

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.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

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.

Isolierung von Maus-Koronar-Endothelzellen

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

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Humane Endothelzellisolierung der Vena saphena und Exposition bei kontrollierter Scherspannung und Dehnung

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

Medizin

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.

Isolation und Kultur der primären Aorten-Endothelzellen von Miniaturschweinen

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 ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Isolierung und Identifizierung vaskulärer Endothelzellen aus unterschiedlichen Fettdepots für Downstream-Anwendungen

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

Isolierung und Kultivierung von primären Hirnperizyten: Ein Verfahren zur Isolierung und Kultivierung von Perizyten aus Rinderhirnkapillaren ohne Kontamination mit Endothelzellen

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...