This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

Fareler ile Aort 1. İzolasyonu Burada anlatılan tüm işlemler Wayne State Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. <ol><li> anestezi makinesinin indüksiyon odasına fare koyun. % 25 taze hava ve% 75 O 2 ile birlikte% 4 izofluran akışını ayarlayın. Özel bir burun konisi tarafından takip indüksiyon odasına aracılığıyla% 25 taze hava ve% 75 O 2 ile birlikte izofluran (indüksiyon için% 4, bakım için 1.0 ±%) solunmasıyla fare anestezisi. NOT: izofluran ile Anestezi% 75 oksijen /% 25 hava veya% 100 oksijen teslim edilebilir. </li><li> Uygun anestezi düzeyi (ayak tutam çekilmesi eksikliği) ulaşılana kadar fareye her 5 dakikada kontrol edin. </li><li> indüksiyon odasının dışında fare alın ve anestezi makinesine bağlı olan özel burun konisi olsa izofluran inhalasyon devam ediyor. Anahtarıizofluran anestezi korumak için% 1 akar. </li><li> o anestezi altında iken, kendi arka yüzünde konumlandırılmış bir ameliyat ped, üzerine fare koymak. bacaklarda sabitlemek için laboratuvar bant kullanın. </li><li> Fare sıcak tutmak için bir ısıtma lambası kullanın. Fare olmaz aşırı ısı böylece fare uygun bir mesafe lamba tutun. </li><li> % 70 etanol ile göğüs püskürtün. </li><li> orta hattan karın açın ve abdominal aorta göstermek için diseksiyon makas kullanın. göğüs boşluğu açın ve kalp ve akciğerler maruz kalmaktadır. </li><li> kan serbest bırakmak için diseksiyon makas ile ortasında bulunan abdominal aorta kesin. </li><li> Heparin 1000 U / mL ihtiva eden 1 mL PBS (25 G iğne ile) 1 ml şırınga doldurun. sol ventrikül heparin 1,000 U / mL içeren PBS enjekte edilir ve aorta serpmek. aorta maruz farelerin sağ tarafına büyük arterlerde forseps ile kalp ve akciğer itin. </li><li> Hızla mikro-di kullanılarak aorta kaldırmakssection forseps ve buz gibi soğuk 1x PBS (steril) koymak, sonra bir laminer hava akımı kaputun içine kabı taşıma. </li><li> Aort bir ucuna 25 G iğne ile donatılmış bir 1 ml şırınga yerleştirin, yavaşça kan çıkarmak için buz soğukluğunda PBS ile aort yıkayın. </li><li> Mümkün olduğunca ekli yağ dokusunun çok küçük ve yan damarları kaldırmak için mikro diseksiyon forseps kullanabilir. </li><li> Hemen büyüme ortamı endotel aort aktarın. </li><li> steril bir neşter bıçak kullanarak 1 mm halkalar şeklinde aorta kesin. Hasat yaklaşık 8 - aort başına 10 yüzük. </li><li> Mikro-diseksiyon makas kullanarak her aort halka açın. </li></ol> 2. Tohum Matrix Aort Bölümleri <ol><li> Kullanılmadığı, katılaşma önlemek için -20 ° C büyüme faktörü azaltılmış matris tutmak zaman. Tam çözülme izin vermek için en az bir gece süreyle 4 ° C&#39;de büyüme faktörü azaltılmış matris koyun. </li><li> en az -20 ° C&#39;de bir 6-yuvalı plaka ve pipet uçları ön soğutma10 dk. Herhangi bir hava baloncuklarının oluşumunu olmayan matris 1 mL plakanın buz ve kat üzerindeki bir 6 yuvalı plaka koyun. 20 dakika matris katılaşmaya izin için bir 37 ° C inkübatör plaka koyun. </li><li> Steril mikro diseksiyon forseps kullanılarak katılaşmış matris üzerine aort parçaları implante edin. endotel dokunmadan lümen tarafı aşağı matris üzerinde parçalarını yerleştirin. matris üzerinde birbirine yakın 4 aort segmentleri - 3 koyun. </li><li> (~ 200 uL), ıslak segmentleri tutmak için yeterli endotelyal hücre büyüme ortamı ilave edin. </li><li> 4 ila 6 saat boyunca% 5 CO2 altında 37 ° C&#39;de inkübe plakası. Günün sonunda, aort segmentleri kaplamak için yeterli miktarda ortam ilave edin. </li><li> Bir faz-kontrast mikroskop altında periyodik olarak çimlenme aort segmenti gözlemleyin. orta düzeyde ve hücre büyümesini her gün kontrol edin. örtülü aort segmentleri tutmak için gerekirse orta ekleyin. </li><li> 4. gününde, yavaşça orta kaldırmak ve aort segmentleri f kaldırmakbüyüyen endotel hücrelerini kesmeden steril iğne kullanılarak matrisi rom. </li><li> Yeni endotel hücre büyüme ortamı 2 ml ilave edilir ve endotel hücreleri 2 matris üzerinde çoğalmaya devam etmesini - 3 gün. </li></ol> Fare Aort Endotel Hücreleri 3. İlk Pasajlanması <ol><li> Kat jelatin (% 0.1) ile birlikte, yeni bir T12.5 şişeye, 30 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Steril 37 ° C PBS ile dikkatlice matris plakaları yıkayın. </li><li> Matris plakalarına nötr proteinazı (50 U / mL) 2 ml ilave edilir ve ara sıra çalkalanarak platform rocker oda sıcaklığında inkübe edin. Hücrelerin büyük çoğunluğu müstakil emin olmak için bir faz-kontrast mikroskop altında hücrelerin kontrol edin. </li><li> Nötr proteinaz inaktif D-Val 2 mL ekleyin. </li><li> 15 ml santrifüj tüpüne dikkatli bir şekilde supernatant toplamak. 2 mL D-Val ile plaka yıkayın ve süpernatant toplamak. </li><li> roo 5 dakika boyunca 900 x g&#39;de hücre süspansiyonu santrifüjm sıcaklığı. </li><li> jelatin ile kaplanmış T12.5 şişede endotelyal hücre büyüme ortamı ve plakanın 4 mL hücre pelletini yeniden askıya. 2 saat boyunca 37 ° C,% 5 CO2 hücreleri inkübe edin. </li><li> orta yerine. % 90 konfluent - bunlar% 85 kadar, 37 ° C&#39;de,% 5 CO2 hücreleri inkübe edin. </li></ol> Fare aort endotel hücreleri 4. pasaj <ol><li> Kat iki yeni T12.5 şişeler jelatin (% 0.1) ile birlikte, 30 dakika süre ile 37 ° C&#39;de inkübe edin. yaklaşık 15 dakika boyunca 37 ° C&#39;de ön ısıtma Tripsin-EDTA (% 0.25 tripsin, PBS içinde 0.02 EDTA) ve steril PBS ile yıkandı. </li><li> Steril 37 ° C PBS ile dikkatlice hücreleri yıkayın. </li><li> Tripsin-EDTA (% 0.25 tripsin, PBS içinde 0.02 EDTA), 0.5 ml ilave edilir ve yaklaşık 1 dakika ya da hücrelerin çoğunluğu boyunca edecek kadar 37 ° C&#39;de inkübe edin. </li><li> sindirim durdurmak için endotelyal hücre büyüme ortamı 2 mL ekleyin. yukarı ve aşağı şişesi birkaç kez içinde hücre süspansiyonu pipetle. Gerekirse, bir hücre kullanabilirhücreleri toplamak için kazıyıcı. </li><li> 15 ml santrifüj tüpüne hücre süspansiyonu toplanır. Oda sıcaklığında 5 dakika boyunca 900 x g&#39;de hücre süspansiyonu santrifüj. </li><li> jelatin ile kaplı 2 T12.5 şişelerinde endotelyal hücre büyüme ortamı ve plakanın 4 mL hücre pelletini yeniden askıya. 2 saat boyunca 37 ° C,% 5 CO2 hücreleri inkübe edin. </li><li> orta yerine. % 90 konfluent - bunlar% 85 kadar, 37 ° C&#39;de,% 5 CO2 hücreleri inkübe edin. </li><li> 3 geçişleri - 2 sonra fare aort endotel hücreleri kullanır. </li></ol> Fare aort endotel hücreleri 5. Karakterizasyonu <ol><li> Yeniden plaka hücreleri. <ol><li> Kat jelatin (% 0.1) ile birlikte, 6 gözlü bir levha, 30 dakika boyunca 37 ° C&#39;de inkübe edin. PBS ile plaka durulayın. </li><li> Ön-ısıtma, yaklaşık 15 dakika boyunca 37 ° C&#39;ye kadar Tripsin-EDTA ve steril PBS. </li><li> Steril PBS ile 3 kez hücreleri yıkayın. hücrelere 0.5 tripsin mL / EDTA ekle, 37 ° C o de ~ 1 dakika boyunca inkübeHücrelerin çoğunda kadar r yuvarlak açın. </li><li> sindirim durdurmak için endotelyal hücre büyüme ortamı 2 mL ekleyin. yukarı ve aşağı şişesi birkaç kez içinde hücre süspansiyonu pipetle. Gerekirse, hücreleri toplamak için bir hücre kazıyıcı kullanın. </li><li> 15 ml santrifüj tüpüne hücre süspansiyonu toplanır. Oda sıcaklığında 5 dakika boyunca 900 x g&#39;de hücre süspansiyonu santrifüj. </li><li> endotelyal hücre büyüme ortamı içinde hücrelerin yeniden askıya, süpernatant atılır. De, 3 x 10 5 / yoğunluğunda jelatin ile kaplanmış 6-çukurlu plaka içindeki hücreleri tohumu. </li><li> Hücreler, kültür yüzey eklemek için bir gece 37 ° C,% 5 CO2 hücreleri inkübe edin. </li></ol></li><li> Dil-ac-LDL bağlama -lectin Alınan ve Ulex. <ol><li> , 1,1-dioktadesil-3,3,3 &#39;deposu 3&#39;- tetramethylindo-karbosiyanin perklorat etiketli -20 ° C&#39;de asetile LDL (Dil-Ac-LDL). boyama önce, buz üzerinde Dil-ac-LDL Çözülme. Stokta Dil-ac-LDL soluma / ml 1 mg&#39;dır. 4 ° C&#39;de FITC işaretli Ulex europaeus agglutinin (Ulex -Lectin, 1 ug / mL) saklayın. Hazır Ulex -Lectin Çözelti 1 mg / mL&#39;dir. Mağaza 4 ° C&#39;de Hoechst 33342. Hazır Hoechst 33342 çözeltisi, 100 ug / mL&#39;dir. </li><li> 10 ug / ml bir nihai konsantrasyon yapmak için kültür ortamı, 1 ml inceltilmiş-Ac-LDL stok solüsyonu 10 uL ekleyin. </li><li> boyama öncesinde, eski orta çıkarın ve yeni ortam ile her bir hücrelerin yıkayın. </li><li> Yeni orta çıkarın ve Dil-ac-LDL boyama orta ekleyin. </li><li> 1 saat boyunca 37 ° C&#39;de,% 5 CO2 hücreleri inkübe edin. </li><li> Steril 1x PBS ile 3 kez hücreleri yıkayın. </li><li> hücrelerin tespit edilmeleri için% 10 formalin 1 ml. yaklaşık 30 dakika boyunca oda sıcaklığında plaka koyun. </li><li> Steril 1x PBS ile 3 kez hücreleri yıkayın. Işığı kaçının. </li><li> Ulex -Lectin 10 uL içeren 1 ml steril 1 x PBS ekleyin. </li><li> Oda temperatu hücreleri inkübe1 saat boyunca karanlıkta yeniden. </li><li> Steril 1x PBS ile 3 kez hücreleri yıkayarak Ulex-lektin çıkarın. </li><li> , 1 ug / ml nihai konsantrasyona kadar Hoechst 33342 ekleme karanlıkta 10 dakika inkübe edilir. </li><li> Dil-Ac-LDL (kırmızı, 576 nm emisyon dalga boyu), Ulex -Lectin (yeşil, 519 nm emisyon dalga boyu) ve Hoechst floresan görüntüleme (mavi, 361 nm emisyon dalga boyu) bir ters flüoresan mikroskop ile. </li></ol></li><li> CD31, VEGFR2&#39;sinin, eNOS için Floresan boyama, VE-kaderin ve calponin. <ol><li> Mağaza FITC-konjüge anti-fare CD31 antikoru, eNOS antikoru, VE-cadherin 4 ° C&#39;de antikoru FITC-konjuge anti-tavşan IgG karanlıkta. -20 ° C&#39;de saklayın VEGFR2 antikoru. </li><li> Steril 1x PBS ile 3 kez hücreleri yıkayın. </li><li> hücrelerin tespit edilmeleri için% 10 formalin 1 ml. yaklaşık 30 dakika boyunca oda sıcaklığında plaka koyun. </li><li> Steril 1x PBS ile 3 kez hücreleri yıkayın. </li><li> CD31 leke için, 1 ml steril 1x ekleyinCD31-FITC antikoru 10 uL içeren PBS ile yıkandı. sırasıyla-VE Cadherin veya primer antikor calponin, leke ya VEGFR2, eNOS,-VE Cadherin veya calponin, VEGFR2&#39;sinin, eNOS 4 mcL içeren steril 1x PBS 1 ml ekleyin. </li><li> Karanlıkta 1 saat boyunca buz üzerinde inkübe hücreleri. </li><li> Steril PBS ile 3 kez hücreler yıkanarak antikor çıkarın. </li><li> VEGFR2 boyanması için, eNOS, VE-cadherin ya calponin FITC-konjuge anti-tavşan IgG, 10 uL içeren steril 1 x 1 mL PBS ilave edin. Karanlıkta 1 saat boyunca buz üzerinde inkübe hücreleri. Steril PBS ile 3 kez hücreler yıkanarak IgG çıkarın. </li><li> , 1 ug / ml nihai konsantrasyona kadar Hoechst 33342 ekleme karanlıkta 10 dakika inkübe edilir. </li><li> CD31, VEGFR2, eNOS floresan görüntüleme VE-cadherin bir ters flüoresan mikroskop ile, calponin (yeşil, 518-535 nm emisyon dalga boyu) ve Hoechst (mavi, 361 nm emisyon dalga boyu). </li></ol></li></ol>

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The authors have nothing to disclose.

Bu çalışma, herhangi bir özel ekipman olmadan bir fare aort hücreleri izole ve kültür endotel için basit bir yöntemi gösterir. immünofloresan boyama hücrelerin çoğunluğu, ikinci geçiş sonra endotel hücreleri olduğunu göstermiştir. Üçüncü geçişine saniyede hücreleri, in vitro ve endotelyal biyolojisi okumak için in vivo deneyler uygun olduğu ileri sürülmektedir. Mevcut Protokolü Önemli Notlar pro...

Vasküler endotelyum, sadece kan ve doku ayıran bir bariyer tabakası, bir 400 metre kare 1 kadar bir yüzey alanı ile damar ağacı boyunca uzanır geniş bir endokrin bezi olarak kabul edilir. endotel refahını damar homeostazının için gereklidir. Disfonksiyonel endotel ateroskleroz, vaskülit ve iskemi / reperfüzyon yaralanması gibi 2-4 dahil olmak üzere çeşitli kardiyovasküler bozuklukların, katılır. Bugüne kadar, bu hastalık ayarlarında yer alan spesifik hücresel ve moleküler mekanizmalar iyi sonucu endotel dağınık anatomik yapısına anlaşılamamıştır. Genetik manipülasyon teknikleri, diğer herhangi bir memeli türlerde daha farelerde geliştirilmektedir, çünkü, fare araştırmaları için önemli bir modeldir. Aort küçük boyutlu enzymat yapar çünkü Ancak, birincil fare aort endotel hücreleri izolasyonu özellikle zor olarak kabul edilirpratik endotel ic sindirimi. Bazı izole ve EC 5-7 gerektirir arındırmak için prosedürler bildirdi. Bu protokolün amacı, herhangi bir özel ekipman kullanmadan fare aorta endotel hücreleri izole ve genişletmek için basit bir yöntem kullanmaktır. Bu protokol, yeni izole edilmiş aort küçük parçalar halinde kesilir ve endotel filizlenmesi için izin vermek için aşağı doğru dönük endotel ile bir matris üzerine tohumlandı. segmentler kaldırıldıktan sonra endotel hücreleri endotel tercih ortamda genişletildi ve iki ya da üç geçişten sonra deneyler için hazırdır. tarif edilen metodun avantajları: 1), tek bir aort hasat endotel hücrelerinin oldukça yüksek sayılar; 2) hücre canlılığı iyi korunmuştur; ve 3) hiçbir özel ekipman veya teknik gereklidir. Bu endotel hücre patofizyolojisinde spesifik hücresel ve moleküler mekanizmaların tanımlanması için etkili bir araç sağlar. pr okuyan ilgilenen olanlar içinHer iki gen knock-out farelerinden alınan imary kültürlenmiş endotel hücreleri, gen knock-fareler ya da bir fare hastalık modeli, bu protokol, çok kullanışlı ve pratik kolaydır.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

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.

Endotel Hücre Çimlenme Spontan endotel hücre, bir fare aort segmentine başladı filizlenme. Fare aortu kademeli bir 4 gün boyunca endotelyal hücre büyüme ortamı içinde daha sonra bir büyüme faktörü azaltılmış matrisi üzerinde büyümeye bırakılmıştır. 4 gün - endotel hücre filizlenmekte genellikle 2 görünür. Fotomikrograflar, 4. günde (Şekil 1) üzerine çekildi. resimlerde gösterildiği gibi, çok sayıda endotel hücreleri uz...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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-and-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.7K Görüntüleme.  Wayne State University. 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.

Video: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

İlköğretim Kemirgen Beyin mikrovasküler endotel hücreleri İzolasyonu

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Nörobilim

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 ...

Biyoloji

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Tıp

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Birincil fare iskelet kas mikrovasküler endotel hücre izolasyon

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Isolation of Embryonic Ventricular Endothelial Cells

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

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

Embriyonik Ventriküler Endotel Hücre İzolasyonu

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

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 of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

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 Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Fare primer karaciğer sinüzoidal endotel hücrelerinin izolasyonu ve karakterizasyonu

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...