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.

Etik bildirimi: Bu çalışmada kullanılan Kan damarları taze köpek kadavra elde edilen artı malzemesi olarak hasat edildi (n = 4), diğer ilişkisiz araştırma (Üniversite 3R politikası) için ötenazi sağlıklı köpeklerden. Anormal kan damarları (intra ve ekstrahepatik portosistemik şantlar, n = 1 adet) Utrecht Üniversitesi Companion Animals Üniversite Kliniği sunulan köpekler sahiplerinin bilgilendirilmiş onam sonra otopsi hasat edilmiştir. 1. İzolasyon ve Kültür İlköğretim Köpek Endotel Hücreleri <ol><li> 2 ml / göz% 0.5 ağırlık / hacim jelatin ile ön-kat 6 oyuklu plakalar, 37 ° C&#39;de% 5 CO2 bulunan nemli bir atmosferde bir kuluçka makinesi içinde 2 saat süre ile bırakın. önce birincil hücrelerin ekim aşırı jelatin çözüm çıkarın. </li><li> Aseptik taze köpek kadavra (Şekil 1A) kan damarı (ler) ilgi (örneğin, aort, vena kava, vena porta) çıkarın. damar sisteminin anatomisini korumakÇevrede kabın her iki ucunda, düz forseps koyarak. endotel hücrelerine zarar görmesini önlemek için forseps ile doku gereksiz manipülasyon kaçının. <ol><li> cerrahi makas ile her iki ucunda kenetlenmiş gemi kesin ve kadavra kaldırmak. buz üzerinde Hank Dengeli Tuz Çözeltisi (HBSS) taşınması. Not: yaklaşık olarak 5 sm bir tekne uzunluğu, bu prosedür için tercih edilir, ancak 1 cm boyutlarında o da kültür için yeterli hücreleri sağlar. </li></ol></li><li> buz soğukluğunda HBSS ile dolu bir Petri kabı, kan damarı aktarın. Cerrahi makas kullanılarak, damar kendisi sağlam (Şekil 1B) tutmak kabın dışından yapışık doku ve yağ çıkarmak. Optimal görünüm için bir kelepçe veya forseps ile kenara çevredeki dokuyu çekin: keserken emin damar her zaman açıkça görünür olduğundan emin olun. <ol><li> Bitişik harfler ile her türlü tekne dalları kapatın (örneğin, 3-0 polyglactin) ve daha sonra bunları kaldırmakcerrahi makas veya neşter ile. </li></ol></li><li> Dikkatle, kavisli bir Halsted sivrisinek forseps ile bir gemi ucunu girmek geminin diğer ucundaki forseps ucu kelepçe ve dışarı (Şekil 1C-G) tamamen içine kadar sonra yavaş yavaş gemiyi tersini, geri çekin. dış artık endotel hücre tabakasını içermektedir. herhangi bir zorluk gemi tersini üzerine karşılaşılırsa, sürtünmeyi azaltmak için tekrar HBSS daldırın. </li><li> Tamamen kapatmak için ve sindirim prosedürü (Şekil 1H) herhangi olmayan endotelyal damar dokusu maruz kalmasını önlemek için geminin her iki ucunda purse-string sütür koyun. ters gemi işlemek için bağ uçlarını kullanın. </li><li> sindirim ve kültür daha sonra yapılırsa, sindirim protokolü (adım 1.7) öncesinde ters gemi cryopreserve. <ol><li> kriyotüpe içinde ters gemi yerleştirin ve hücre kültür ortamı dondurma ile doldurun. Bir dondurma co kullanarak aşağı -80 ° C&#39;ye kadar Freezentainer. -80 ° C&#39;de saklayın gemiler bir hafta içinde kullanılacak ise. Uzun süreli depolama, -180 ° C&#39;de yer cryovials için. AT izolasyon gerçekleştirirken, bir su banyosu (37 ° C) hızla cryovials çözülme hemen buz soğukluğunda HBSS yerleştirin. 1.7 belirtildiği gibi devam edin. Not: izolasyondan sonra EC toplam verimi sayesinde dondurma işlemi sırasında canlılığı kaybına daha düşük olacağı dikkate alacaktır. </li></ol></li><li> 50 ml&#39;lik bir tüp gemi aktarın ve (Şekil 1I) (çözülme durumunda veya artık dondurma orta) eritrositler kaldırmak HBSS iki kez yıkayın. </li><li> Aralıklı yumuşak olan 37 ° C &#39;de 1 saat boyunca köpek Endotel Hücrelerinde 30 mi kolajenaz tip II (0.15 U / ml) ve dispase (0.15 U / ml) içeren bir çözeltiye Gelişim Ortamı (CECGM) olan bir 50 ml tüp içinde damar Digest çalkalama. </li><li> g tüpten gemi çıkarın ve x 250 5 dakika boyunca hücre süspansiyonu santrifüj. </li><li> CECGM a hücre pelletinind tohum 2 ml hücre süspansiyonu oyuk başına (1-3 kuyu damar boyutuna bağlı olarak). Kültür hava içinde% 5 CO2 ile nemli bir atmosferde 37 ° C &#39;de hücreleri ve haftada iki kez kültür ortamı değiştirin. </li><li> % 80 ulaşıldığında - 70 bir confluency Passage hücreleri. <ol><li> Ölü ya da olmayan bağlanmış hücreler uzaklaştırılmıştır önceden ısıtılmış HBSS hücreleri yıkayın. Her bir oyuğa 200 ul rekombinant hücre-ayrılma enzimi (1x) ilave edin ve 5 dakika boyunca ya da tüm hücrelerin müstakil kadar tekrar inkübatör yerleştirin. </li><li> 15 ml&#39;lik bir tüpe transfer hücreleri ve% 10 cenin buzağı serumu (FCS) içeren kültür ortamı 10 ml (CEGM) eklenerek tripsinizasyon durdurun. 250 x g hızında 5 dakika süreyle santrifüj. Jelatin önceden kaplanmış plaka veya şişede kültür devam edin. </li></ol></li></ol> 2. Karakterizasyonu <ol><li> Kültür Köpek aort endotel hücreleri (CnAOECs) ve bir mikroskop ile haftada iki kez birincil ve ticari hücre hatlarının morfolojisi karşılaştırın. NOT: TO, tipik&gt;% 70 konfluansa ulaştığında iyi görülmektedir yamalar EC desen büyüyen. <ol><li> % 0.5 ağırlık / hacim jelatin önceden kaplanmış T75 şişesinin üzerine CECGM Kültür CnAOECs. haftada bir kez Passage hücreleri confluency 70 -% 80. <ol><li> geçirilmesi için önceden ısıtılmış bir kez HBSS ile yıkama hücreleri, 1 ml yeniden birleştirici hücre ayrışma enzimi (1x) ekleyin. 5 dakika boyunca veya tüm hücreler müstakil kadar inkübatör geri şişeyi yerleştirin. % 10 FCS içeren 10 ml kültür ortamı (CEGM) eklenerek tripsin inaktive. 250 x g&#39;de 5 dakika boyunca bir hücre süspansiyonu santrifüj süpernatan atılır ve 1 ml kültür ortamında tekrar süspansiyon hücreleri. </li><li> Hücre süspansiyonu, 10 ul bir kısım almak ve 1 seyreltin:% 0.4 tripan mavisi ile 1. Otomatik hücre sayıcı kullanarak hücre sayımı ve yeni T75 şişesi içinde 4.0 x 10 5 canlı hücreleri plaka. % 5 CO2 ile nemli bir atmosferde 37 ° C&#39;de bir şişeye ve kültür önceden ısıtılmış CECGM 10 ml ekleyin. </li></ol></li></ol></li><li> Kültür CaPECs ve CnAOECs geçişi 1 RNA izole edin. <ol><li> Bir pelet gibi en az 1 x 10 3 hücreleri toplamak örnek hazırlama reaktifi (SPR) 20 ul ekle ve hücreleri lize etmek için 1 dakika için inkübe edilir. İnkübasyondan sonra, dikkatli bir şekilde -70 ° C&#39;de, toplam RNA ve mağaza ihtiva eden hücre lizatı toplar. </li></ol></li><li> Üreticinin talimatları izlenerek bir cDNA sentez kiti (örneğin, iScript) kullanılarak cDNA&#39;ya mRNA dönüştürün. qPCR gerçekleştirmek için hazır olana kadar uzun süre için 4 ° C&#39;de bir hafta kadar ya da -20 ° C&#39;de saklayın cDNA. </li><li> endotel hücre kaynağını teyit etmek için endotelyal işaretleyici CD31 gen ifadesini ölçün. MIQE Özetleyerek yönergeleri 10 kullanılarak deney düzeneği ve kantifikasyonunu gerçekleştirin. Normalleşmesi için, referans genler GAPDH, RPS19 ve B2MG 11 ifadesini ölçmek. <ol><li> nükleaz içermeyen su elde edilen cDNA bir 10-kat seyreltme hazırlayın. </li><li> hazırlamakStandart hattı için bir araya getirilmiş cDNA örnekleri bir 4-kat seyreltme ve şablon olmayan kontrol olarak nükleaz serbest su kullanın. qPCR reaksiyonları için 50 kat, toplam seyreltme ulaşmak için numune beş kez seyreltilir. 4 ul cDNA ve geri ve ileri doğru primer (Tablo 1) 20 pmol ile karıştırılmış fluorofor 6 ul kullanılarak 384 oyuklu bir formatta iki kez pipetle 10 ul reaksiyonları. </li><li> denatürasyon için 95 ° C de 10 saniye 39 döngü, ardından Taq polimeraz aktivasyonu için 5 dakika, tavlama ve uzama için Tm 30 saniye boyunca 95 ° C&#39;ye kadar bir program ayarlama. Her bir primer grubu için Tm Tablo 1 &#39;de gösterilmiştir. </li><li> erime eğrisi gerçekleştirin yükseltilir tek bir ürün sağlamak için her çalıştırmak aşağıdaki analiz eder. Referans genlerin ortalama bağıl miktarda kullanarak numunelerin ifade düzeylerini normale ve reaksiyon verimi% 95 ile% 105 arasında ise ACt hesaplayın. </li></ol></li><li> Bir Angi EC işlevselliğini değerlendirmekogenesis deneyi. <ol><li> önceden soğutulmuş bir anjiyojenez sürgünün her çukuruna hücre dışı matrisin 10 ul ilave edilip iyice yüzeyini kaplayan bir pipet ucu ile yayılır. buz üzerinde hücre dışı matris tutmak ve jele hava kabarcıklarının giriş önlemek. 30 dakika boyunca% 5 CO2 ile 37 ° C &#39;de, bir kuluçka makinesi içinde suda ıslatılmış kağıt havlu ile bir Petri kabındaki slayt koyarak jel katılaşmaya. </li><li> 1.0 x 10 4 birincil ekle EC 50 ul Endotel Gelişim Ortamı ile sabitleştirilir. % 5 CO2 ile 37 ° C &#39;de 6 saat süre ile slayt inkübe edin. </li><li> 6 saat sonra görüntüdeki tüm kuyu içerdiğinden emin yapma, 20X büyütme ile fotoğraf çekmek. </li></ol></li></ol>

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The authors have no competing financial interests.

Köpek EC odaklanan çalışmalarda CnAOEC birincil hat köpek 3, 12, 13 endotel soy modellemek için kullanılır. İnsan çalışmalarında, HUVEC kültürü hala altın standart olarak kabul edilmektedir. Açıkçası, göbek kordonu elde edilen EC üzerinde sadece odaklanan Kardiyovasküler araştırmalarda bir firma kısıtlama olduğunu. Endotel hücreleri arteriovenöz özelliklerinin belirlenmesiyle belirli bir gen ifade desen var. Doğum sonrası damarlarda bu farklılıkların hesaba katmak için bi...

Köpekler kardiyovasküler hastalık araştırmaları için büyük bir hayvan modeli olarak kullanılmaktadır ve aynı zamanda doğuştan (genetik) vasküler anormallikler 1, 2 muzdarip olabilir. Ticari endotel hücre hatları genellikle endotel hücre (EC) işlevselliğini değerlendirmek için kullanılan bu hastalıkların incelenmesi. köpekler için köpek aortundan alınan (CnAOEC) mevcuttur ticari bir endotelyal hücre hattı vardır. Bu hücre hattı çoğunlukla kontrol, normal EC 3-5 olarak çalışmalarda kullanılmaktadır. kan dolaşım araştırmada en yaygın olarak kullanılan endotel hücre çizgileri sırasıyla insan göbek kordonu damar ve arter türetilen insan Göbek Daman Endotel Hücreleri (HUVECs) ve İnsan Umbilikal Arter Endotel Hücreleri (HUAECs) vardır. HUVECler 1980&#39;lerden 6 yılından bu yana damar araştırmalarda altın standart olarak kullanılmıştır. Onlar endotel fonksiyonu ve hastalık adaptasyonunu incelemek için klasik model sistem olarak kabul edilmektedir. Farklı kan damarları izole endotel hücreleri appearanc değişirE ve mikroçevresinin 7 genetik arka plan ve maruz kalma nedeniyle işlevsellik. Buna ek olarak, HUVECler ve HUAECs, göbek kordonu şartlarına göre tamamen taklit yetişkin kan damarlarının onlar ve hastalığa yanıt maruz olmayabilir gelişimsel vasküler yapı elde edilir. Bu nedenle, genel olarak kardiyovasküler hastalık için HUVEC bulunan sonuçları ve HUAECs tercüme yetersizdir. adaptasyon ve yetişkin EC davranışını incelerken, ilgilenilen damarın birincil EC daha doğrudan bir yaklaşım olarak kullanılmalıdır. bu hücrelerin izole edilmesi için, çeşitli yöntemler bildirilmiştir. Ayrıca, HUVEC için kullanılan bir çok tarif edilen yöntem, enzimatik sindirim çözeltisi 8 kap yıkama edilir. Bu çoğu zaman, düz kas hücreleri, fibroblastlar ve 9 olmayan EC ile kontaminasyon sonuçlanır. izolasyon için yaygın olarak kullanılan bir yöntem, Floresan-takiben kıyılmış damar doku enzimatik sindirimi olanFarklılaşma (CD) 31 7, 8. FACS ayıklama endotelyal hücre belirteci küme ve daha sonra, hücre kültürü göre (FACS) aktif hücre hücrelerin nispeten büyük miktarlarda gerektirir ve bu nedenle ince kan damarlarından endotelyumun izolasyon için uygun değildir. Bu nedenle yüksek saflıkta çeşitli köpek kan damarlarından saf endotel hücre popülasyonunu izole etmek için yeni bir sağlam bir yöntem geliştirmeyi amaçladık. Yeni izolasyon yönteminin etkinliğini test etmek için, izole ve büyük ve küçük her ikisi de farklı köpek arterler ve venler, saf Canine Primer endotel hücre (CapeC) kültürleri elde etti. Bu yöntem aynı zamanda doğuştan içi veya dışı hepatik portosistemik şant, köpekler 2 ortak bir hastalık olarak hastalıklı ve / veya anormal damarlardan kaynaklanan endotel hücrelerinin kültürü sağlar. kabın en int olarak kalmaktadır, bu yöntem, vasküler düz kas hücreleri gibi aynı geminin alakalı ek hücre tiplerinin izolasyonu sağlarİşlem sırasında hareket ederler.

<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.

Farklı kan damarları başarıyla açıklanan izolasyon protokolü (Şekil 2) tabi tutuldu. Teşrih ve sağlıklı köpeklerden aort, vena kava, vena porta ve koroner arter ters mümkün olmuştur (her köpekten tüm damarları, n = 4). Aynı yaklaşım EC iki doğumsal portosistemik şant (ekstrahepatik ve intrahepatik, n = 1 adet) izole edilmiştir ile. Aort kolayca ters olmasına rağmen, aort segmentleri abdominal aorta daha zorlu idi. göğüs segmentlerde aort sin...

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.

Köpek arter ve venlerin Primer Endotel Hücreleri İzolasyon ve Kültür

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

Research

JoVE Journal

Medicine

11.2K Görüntüleme.  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.

Kapak Endotel Hücreleri İzolasyon

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

Biyoloji

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.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

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.

Fare Direnç arterlerden mikrovasküler endotel Tüpler İzolasyonu

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.

Primer Fare Akciğer Endotel Hücrelerinin İzolasyonu

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

İmmünoloji ve Enfeksiyon

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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.

Fare Koroner Endotel Hücreleri İzolasyon

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.

İnsan safen ven endotel hücre izolasyonu ve kontrollü kayma stresi ve gerilme seviyelerine maruz kalma

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

Tıp

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.

Minyatür Domuzlardan Birincil Aort Endotel Hücrelerinin İzolasyon ve Kültürü

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.

Vasküler Endotel Hücrelerinin Aşağı Akış Uygulamaları için Farklı Yağ Depolarından İzolasyonu ve Tanımlanması

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://www.jove.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.

Primer Beyin Perisitlerinin İzolasyonu ve Kültürü: Endotel Hücre Kontaminasyonu Olmadan Sığır Beyin Kılcal Damarlarından Perisitleri İzole Etme ve Kültür Prosedürü

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