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.

Etica dichiarazione: I vasi sanguigni utilizzati in questo studio sono state raccolte come materiale in eccesso ottenuti da cadaveri freschi canino (n = 4) da cani sani eutanasia per altre ricerche non correlate (politica Università 3R). vasi sanguigni aberranti (shunt porto intra- e extra-epatiche, n = 1 ciascuno) sono state raccolte post mortem dopo consenso informato dei proprietari di cani presentati alla clinica universitaria per Animali da Compagnia di Università di Utrecht. 1. Isolamento e la coltura di cellule primarie Canine endoteliali <ol><li> Pre-coat 6 pozzetti con 2 ml / pozzetto 0,5% w / v di gelatina e lasciare per 2 ore in un incubatore con atmosfera umidificata al 5% di CO 2 a 37 ° C. Rimuovere soluzione di gelatina in eccesso prima semina delle cellule primarie. </li><li> Asetticamente rimuovere il vaso sanguigno (s) di interesse (ad esempio, aorta, vena cava, vena porta) a partire da un fresco canino cadavere (Figura 1A). Mantenere l&#39;anatomia del sistema naveponendo pinze diritte su entrambe le estremità del vaso di interesse. Evitare manipolazioni inutili del tessuto con una pinza per evitare danni alle cellule endoteliali. <ol><li> Tagliare la nave bloccato su entrambe le estremità con le forbici chirurgiche e togliere dal cadavere. Trasporti in soluzione salina bilanciata di Hank (HBSS) su ghiaccio. NOTA: Una lunghezza imbarcazione di circa 5 cm è preferito per questa procedura, ma quelli che misurano 1 cm fornirà anche abbastanza cellule per la cultura. </li></ol></li><li> Trasferire il vaso sanguigno di una capsula di Petri riempite con HBSS ghiacciata. Utilizzando le forbici chirurgiche, rimuovere eventuali tessuti aderenti e grasso dall&#39;esterno del recipiente, mantenendo il recipiente stesso intatta (Figura 1B). Assicurarsi che il vaso è ben visibile in ogni momento durante il taglio: tirare a parte il tessuto circostante con un morsetto o pinze per la visualizzazione ottimale. <ol><li> Chiudere tutti i rami della nave con legature (ad esempio, Polyglactin 3-0) e, successivamente, rimuoverlicon le forbici chirurgiche o un bisturi. </li></ol></li><li> Immettere cautela un&#39;estremità recipiente con curve pinza zanzara Halsted, bloccare la punta della pinza all&#39;altra estremità della nave e quindi ritrarre, invertendo lentamente il serbatoio finché è completamente esterno (Figura 1C-G). L&#39;esterno ora consiste dello strato di cellule endoteliali. Se si incontra difficoltà sulla invertendo il vaso, immergerlo in HBSS nuovamente per ridurre l&#39;attrito. </li><li> Posizionare suture a borsa di entrambe le estremità della nave per chiuderla completamente e per evitare l&#39;esposizione di qualsiasi tessuto vascolare non-endoteliale alla procedura di digestione (Figura 1H). Utilizzare le estremità legatura di manipolare la nave rovesciata. </li><li> Se la digestione e la cultura viene eseguita in seguito, cryopreserve la nave rovesciata prima del protocollo di digestione (passo 1,7). <ol><li> Posizionare il vaso rovesciato in una provetta Microbank ™ e riempire con colture cellulari congelamento media. Antigelo fino a -80 ° C con una co congelamentontainer. Conservare a -80 ° C se i vasi devono essere utilizzati entro una settimana. Per la conservazione a lungo termine, luogo cryovials a -180 ° C. Quando si esegue l&#39;isolamento CE, scongelare i cryovials rapidamente in un bagno d&#39;acqua (37 ° C) e collocare immediatamente in ghiaccio freddo HBSS. Procedere come indicato in 1.7. NOTA: Prendere in considerazione che la resa totale di EC dopo l&#39;isolamento sarà inferiore a causa della perdita di vitalità durante il processo di congelamento. </li></ol></li><li> Trasferire la nave verso un tubo da 50 ml e lavare due volte in HBSS per rimuovere gli eritrociti (o medio congelamento residua in caso di scongelamento) (Figura 1I). </li><li> Digerire il vaso in un tubo da 50 ml con una soluzione di 30 ml di collagenasi di tipo II (0,15 U / ml) e dispasi (0,15 U / ml) in cellule Canine endoteliali Growth Medium (CECGM) per 1 ora a 37 ° C con intermittente delicato agitazione. </li><li> Rimuovere il recipiente dal tubo e centrifugare la sospensione cellulare per 5 minuti a 250 x g. </li><li> Risospendere il pellet di cellule in un CECGMND seme 2 sospensione cellulare ml per bene (1 - 3 pozzi a seconda delle dimensioni nave). Coltivare le cellule a 37 ° C in atmosfera umidificata con 5% di CO 2 in aria e cambiare il terreno di coltura due volte a settimana. </li><li> Passaggio delle cellule quando una confluenza di 70 - si raggiunge l&#39;80%. <ol><li> Lavare le cellule con HBSS preriscaldata per rimuovere le cellule morte o non iscritti. Aggiungere 200 ml ricombinante enzima cellulare-dissociazione (1x) per ciascun bene e mettere di nuovo in incubatrice per 5 minuti o fino a quando tutte le cellule sono staccati. </li><li> Trasferire le cellule in una provetta da 15 ml e fermare tripsinizzazione con l&#39;aggiunta di 10 ml di terreni di coltura (CEGM) di cui 10% siero fetale di vitello (FCS). Centrifugare per 5 min a 250 x g. Continuare la cultura in un piatto di gelatina pre-verniciato o pallone. </li></ol></li></ol> 2. Caratterizzazione <ol><li> Culture cellule endoteliali aortiche Canine (CnAOECs) e confrontare la morfologia delle linee cellulari primarie e commerciale due volte alla settimana con un microscopio. NOTA: Tegli tipico modello di crescita di cellule endoteliali in patch può essere meglio osservato quando viene raggiunta una confluenza di&gt; 70%. <ol><li> Culture CnAOECs in CECGM su una beuta T75 0,5% w / v di gelatina prerivestita. cellule Passage una volta alla settimana quando la confluenza è del 70 - 80%. <ol><li> Per passaging, lavare le cellule una volta con HBSS preriscaldata e aggiungere 1 ml ricombinante cella-dissociazione enzimatica (1x). Porre il pallone torna in incubatrice per 5 minuti o fino a quando tutte le cellule sono staccati. Inattivare tripsina con l&#39;aggiunta di mezzo di coltura 10 ml (CEGM) incluso 10% FCS. Centrifugare la sospensione cellulare per 5 minuti a 250 xg, scartare il surnatante e risospendere le cellule in terreno di coltura 1 ml. </li><li> Prendete una aliquota di 10 microlitri di sospensione cellulare e diluire 1: 1 con il 0,4% trypan blu. Contare le cellule utilizzando un contatore di cellule automatizzato, e piastra out 4.0 x 10 5 cellule vitali in un pallone nuovo T75. Aggiungere 10 ml di pre-riscaldato CECGM al pallone e coltura a 37 ° C in atmosfera umidificata con 5% di CO 2. </li></ol></li></ol></li><li> Isolare RNA dal passaggio 1 delle CaPECs coltivate e CnAOECs. <ol><li> Raccogliere almeno 1 x 10 3 cellule come pellet, aggiungere 20 ml di preparazione del campione reagente (SPR) e incubare per 1 min per lisare le cellule. Dopo l&#39;incubazione, raccogliere con attenzione il lisato cellulare contenente RNA totale e conservare a -70 ° C. </li></ol></li><li> Convertire mRNA in cDNA utilizzando un kit di sintesi di cDNA (ad esempio, iScript) seguendo le istruzioni del produttore. Conservare cDNA a 4 ° C fino a una settimana, oa -20 ° C per il lungo termine fino al momento di eseguire la qPCR. </li><li> Misurare l&#39;espressione del gene del CD31 marcatore endoteliale per confermare l&#39;origine delle cellule endoteliali. Eseguire l&#39;impostazione sperimentale e quantificazione utilizzando le linee guida precis MIQE 10. Per la normalizzazione, misurare l&#39;espressione di geni di riferimento GAPDH, RPS19, e B2MG 11. <ol><li> Preparare una diluizione di 10 volte del cDNA ottenuto in acqua priva di nucleasi. </li><li> Preparareuna diluizione di 4 volte da campioni di cDNA pool per la linea standard e utilizzare l&#39;acqua priva di nucleasi come un controllo non-modello. Diluire i campioni cinque volte per raggiungere una diluizione totale di 50 volte per le reazioni qPCR. Pipettare 10 microlitri reazioni in duplicato in un formato da 384 pozzetti con 4 ml di cDNA e 6 ml di fluoroforo mescolato con 20 pmol di retromarcia e primer forward (Tabella 1). </li><li> Impostare il programma a 95 ° C per 5 minuti per l&#39;attivazione polimerasi Taq, seguita da 39 cicli di 10 secondi a 95 ° C per la denaturazione, e 30 sec a Tm per la ricottura e l&#39;allungamento. La Tm di ogni primer è mostrato nella Tabella 1. </li><li> Eseguire la fusione curva di analisi eseguite dopo ogni corsa al fine di garantire un solo prodotto è amplificato. Normalizzare i livelli di espressione dei campioni con la quantità media relativa dei geni di riferimento, e calcolare il ΔCt se l&#39;efficienza reazione è compresa tra il 95% e il 105%. </li></ol></li><li> Valutare la funzionalità CE con un Angisaggio ogenesis. <ol><li> Aggiungere 10 ml di matrice extracellulare a ciascun pozzetto di una slitta pre-raffreddata angiogenesi e diffondere con un puntale per coprire la superficie del pozzetto. Mantenere la matrice extracellulare su ghiaccio ed evitare l&#39;introduzione di bolle d&#39;aria nel gel. Solidificare il gel ponendo il vetrino in una capsula di Petri con acqua imbevuto asciugamani di carta in un incubatore a 37 ° C con 5% di CO 2 per 30 min. </li><li> Aggiungere 1,0 x 10 4 primario EC in 50 microlitri Endothelial Growth medio per pozzetto. Incubare il vetrino per 6 ore a 37 ° C con 5% di CO 2. </li><li> Dopo 6 ore scattare fotografie con un ingrandimento 20x, avendo cura di includere l&#39;intera bene nell&#39;immagine. </li></ol></li></ol>

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The authors have no competing financial interests.

In studi incentrati sul canino EC linea primaria CnAOEC viene utilizzato per modellare le linee endoteliali del cane 3, 12, 13. Negli studi umani, la cultura HUVEC è ancora considerato il gold standard. Chiaramente, concentrandosi solo sulla EC derivate da cordone ombelicale è una restrizione fermo nella ricerca cardiovascolare. Le cellule endoteliali hanno una specifica pattern di espressione genica che determina le specifiche artero-venosa. Per tenere conto di queste differenze di vasi postnatale presenti...

I cani vengono utilizzati come grande modello animale per la ricerca sulle malattie cardiovascolari e possono anche soffrire di innata (genetici) anomalie vascolari 1, 2. Per studiare queste malattie linee di cellule endoteliali commerciali sono spesso utilizzati per valutare la funzionalità delle cellule endoteliali (CE). Per i cani c&#39;è una linea commerciale delle cellule endoteliali a disposizione (CnAOEC), derivato da dell&#39;aorta canino. Questa linea cellulare è usato soprattutto in studi come normale controllo EC 3-5. Nella ricerca cardiovascolare umano linee di cellule endoteliali più comunemente usati sono cellule ombelicale Vena endoteliali (HUVEC) e ombelicale umano Arteria cellule endoteliali (HUAECs) derivate dalla vena umana cordone ombelicale e delle arterie, rispettivamente. HUVECs sono stati usati come il golden standard nel campo della ricerca vascolare dal 1980 6. Essi sono considerati essere il sistema modello classico per studiare la funzione endoteliale e adattamento malattia. Le cellule endoteliali isolate da diversi vasi sanguigni variano in appearancE e funzionalità a causa di background genetico e l&#39;esposizione al microambiente 7. Inoltre, HUVEC e HUAECs derivano dal cordone ombelicale, una struttura vascolare sviluppo che potrebbero non pienamente mimici vasi sanguigni adulto rispetto alle condizioni a cui sono esposti e la risposta alla malattia. Quindi, traducendo Risultati trovati in HUVECs e HUAECs per le malattie cardiovascolari, in generale, è inadeguata. Quando si studia l&#39;adattamento e il comportamento degli adulti EC, EC primari dal vaso di interesse dovrebbero essere usati come un approccio più diretto. Per isolare queste cellule, sono stati riportati diversi metodi. Un metodo ampiamente descritto, che è anche usato per HUVEC, sta eliminando il recipiente con una soluzione digestione enzimatica 8. Questo si traduce spesso in contaminazione con non-EC come le cellule muscolari lisce e fibroblasti 9. Un altro metodo frequentemente utilizzato per l&#39;isolamento è la digestione enzimatica del tessuto nave tritato seguito da fluorescence-cellule attivate (FACS) basato su endoteliali Cluster indicatore di cella di differenziazione (CD) 31 7, 8. FACS ordinamento e successiva coltura cellulare richiede quantità relativamente elevate di cellule e non è quindi adatto per l&#39;isolamento di endotelio da piccoli vasi sanguigni. Abbiamo quindi finalizzata allo sviluppo di un nuovo metodo robusto per isolare una popolazione di cellule endoteliali puro da vari vasi sanguigni canine con elevata purezza. Per verificare l&#39;efficacia del nuovo metodo di isolamento, abbiamo isolato e ottenuto colture pure Canine endoteliali primarie cellulare (CAPEC) da diverse arterie e vene canini, grandi e piccoli. Questo metodo consente anche la cultura di cellule endoteliali provenienti da malati e / o vasi aberranti come innate shunt porto intra o extra-epatiche, una malattia comune nei cani 2. Il metodo permette l&#39;isolamento di altri tipi di cellule competenti della stessa nave, come le cellule muscolari lisce vascolari poiché la maggior parte della nave rimane intagire durante la procedura.

<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.

Diversi vasi sanguigni sono stati sottoposti con successo al protocollo descritto isolamento (Figura 2). Era possibile sezionare e invertire aorta, vena cava, vena porta, e l&#39;arteria coronaria da cani sani (tutte le navi da ciascun cane, n = 4). Con le stesse EC approccio sono stati isolati da due shunt porto congenite (extraepatica e intraepatica, n = 1 ciascuno). Anche se dell&#39;aorta era facilmente invertita, segmenti dell&#39;aorta toracica sono stati più impe...

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.

Isolamento e coltura di cellule endoteliali primarie dalle arterie e vene Canine

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 Views.  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.

Video: 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.

Isolamento di cellule endoteliali valvolare

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

Ricerca

Biologia

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 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.

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

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.

Isolamento delle cellule endoteliali polmonari primarie di topo

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

Immunologia e infezioni

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 ...

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.

Vena safena umana Isolamento delle cellule endoteliali ed esposizione a livelli controllati di stress da taglio e allungamento

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

Medicina

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 ...

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.

Isolamento e identificazione di cellule endoteliali vascolari da depositi adiposi distinti per applicazioni a valle

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.

Isolamento e coltura di periciti cerebrali primari: una procedura per isolare e coltivare periciti da capillari cerebrali bovini senza contaminazione delle cellule endoteliali

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