Bu araştırma, NSF KARİYER ödülü, Hartwell Vakfı ve Amerikan Kalp Derneği (# 0830384N) tarafından desteklenmektedir.

1. Hazırlık <ol><li> Kapalı bir enstrüman Otoklav aşağıdaki öğeleri tepsi: <ol><li> Tırtıklı doku forseps - broşür doku taşıma için </li><li> Doku makas (8 cm) - broşür doku ve yaprakçıkların kırpma </li><li> Pamuk Swablar - broşür veya tüberkülü endotel tabakası izole için </li></ol></li><li> Steril kollajenaz çözüm olun <ol><li> 4.0 gram toz DMEM 250 mL 18 M su ekleyin. </li><li> 1.11 gram sodyum bikarbonat ekleyin. </li><li> Kollajenaz, 180.000 adet (600 U / ml) ekleyin. </li><li> % 1 (3ml) Penisilin / Streptomisin ekleyin. </li><li> PH 7.2 &#39;ye çözüm ayarlayın. </li><li> 270 ml çözüm getirin. </li><li> 0.2 mikron filtre geçerek çözüm sterilize edin. </li><li> % 10 (30 ml) steril fetal sığır serumu ekleyin. </li></ol></li><li> Steril endotel hücre orta olun <ol><li> 6.7 gram toz DMEM 400 mL 18 M su ekleyin. </li><li> 1.85 gram sodyum bikarbonat ekleyin. </li><li> (50 U / ml) 25.000 ünite heparin ekleyin. </li><li> % 1 (5 mL) Penisilin / Streptomisin ekleyin. </li><li> PH 7.2 &#39;ye çözüm ayarlayın. </li><li> 450 mL çözüm getirin. </li><li> 0.2 mikron filtre geçerek çözüm sterilize edin. </li><li> % 10 (50 ml) steril fetal sığır serumu ekleyin. </li></ol></li><li> Steril interstisyel hücre orta olun <ol><li> 13.37 gram toz DMEM 800 mL 18 M su ekleyin. </li><li> 3.7 gram sodyum bikarbonat ekleyin. </li><li> % 1 (10 ml) Penisilin / Streptomisin ekleyin. </li><li> PH 7.2 &#39;ye çözüm ayarlayın. </li><li> 900 mL çözüm getirin. </li><li> 0.2 mikron filtre geçerek çözüm sterilize edin. </li><li> % 10 (100 ml) steril sığır serumu ekleyin. </li></ol></li></ol> 2. Kalp Vana Broşürler izolasyonu <ol><li> Aort kökü kurban sonra kalp ve hemen aseptik Vergi. </li><li> Kan aort kökü soğuk steril DPBS ile iyice durulayın. VEC ölüm ve bakteriyel kontaminasyon sınırlamak için tüm kan bileşenleri en kısa sürede kaldırmak için zorunludur. Antibiyotikler ve antimikotikleri VEC potansiyel zararlı olduğundan bu aşamada önerilmemektedir. </li><li> Doğrudan, 12 mL soğuk DPBS dolu steril 15 ml konik tüp kök ve yerden kapakçıkların (3 valf başına) yalıtın. Taze DPBS enkaz ve dolum kaldırmak için birkaç kez çalkalayınız. </li><li> Buz üzerinde laboratuar geri taşınması. </li><li> Laboratuarda varışta, steril kaputun altındaki doku ile kap yerleştirin. </li></ol> 3. Endotel Katman İzolasyonu <ol><li> Valf başına soğuk kollajenaz çözüm (3 broşürler) 3ml steril bir 35mm çanak doldurun. </li><li> Kollajenaz çözümü ile dolu çanak içine 15 ml tüp üç broşürler yerleştirin. </li><li> 37 5-10 dakika doku inkübe ° C </li><li> Endotel tabakası broşürün yüzeyine kuru steril bir çubukla çevirerek yavaşça çıkarın. Kayma uygulanan dönme yönü ve miktarı numune saflık için kritik öneme sahiptir. Çubukla dönme elinizi doğrusal hareket kontrollü bir kesme oluşturmak için bir ters yönde olmalıdır. Bu kayma, doku endotel hücreleri ne asansörleri. Kuvvet uygulanan miktar doku direnci hissediyorum ama, bazal membran nüfuz için yeterli olmalıdır. </li><li> Bazen, dab kollajenaz çözüm içindeki çubukla ucu lifleri hücreleri çıkarmak için. Sürüntü tamamlandıktan sonra, endotel tabakasının doku öncesine göre biraz daha yumuşak hissediyorum gerekir. </li><li> Hücre süspansiyonu / kollajenaz toplayın ve yeni bir 15 mL steril tüp transferi. </li><li> Pelet için 5 dakika herhangi bir izole hücreler için tüpleri 1000 rpm&#39;de santrifüj ve süpernatant aspirat. Iyi interstisyel hücreler izole Eğer bu hücreler santrifüj edilirken, bu protokol yapmak. </li><li> 3 ml, 15 ml tüpler endotel porcine orta, ikinci kez santrifüj ve aspirat medya. Bu ikinci santrifüj ucu lifleri gibi bazı istenmeyen malzeme filtre yardımcı olur. </li><li> Santrifüje endotel hücreleri, kolajen ile bir ön kaplı T-25 balonuna (kullanım santrifüj tüp başına 1 balon) 5 ml endotel porcine orta ve plaka hücrelerin yeniden askıya. </li><li> Hücrelerin endotel orta değiştirmeden önce en az 2-3 gün büyümesine izin. Bu hücrelerin kurtarmak ve izolasyon işlemi oldukça sert ve hücre verimi düşük olabilir bu yana bölmek yardımcı olur. Temas inhibisyonu hücre dönüşümün yol açabileceği için izdiham yakın geçiş hücreleri için çok önemlidir. </li></ol> 4. Side Özel İzolasyon 60 mm Kültür Bulaşık hazırlanması <ol><li> Alüminyum folyo ile (Line 60 mm cam petri kaplarına leafle başına 2 cam yemeklerit). Alüminyum folyo, cam petri diğer izolasyonların yeniden böylece, parafin giderilmesine yardımcı olur. </li><li> Yaklaşık yarısı dolu, bulaşıkları içinde parafin boncuklar yerleştirin ve otoklav için kapak. </li><li> Otoklav tamamlandı ve parafin eritilmiş sonra, serin bir düz yüzeye çanak topluluk taşıyın. Parafin soğudukça, sertleşecek ve iğne destek olacak bir katman oluşturun. </li><li> 30 dakika sonra, sterilize çanak broşür doku hareketsiz için bir izolasyon odası olarak kullanılan olabilir. </li></ol> 5. Side Özel Endotel Layer izolasyonu <ol><li> Broşürler tarafında belirli bir izolasyonu için hazırlanan 60mm kültür kaplarına 15 mL tüpler ve yerden çıkarın. </li><li> Broşürün ventricularis yan fibrosa yan maruz bırakarak parafin yüzey üzerine yüzü aşağı bakacak şekilde işleyebilirsiniz. Endotel tabakasının açığa çıkarmak için broşürün kenarlarına iğne. </li><li> 37 her biri için (yukarı doğru bakan) endotel yüzeyinde bir kaç damla soğuk kollajenaz koyun ve 5-10 dakika doku inkübe ° C </li><li> Daha önce olduğu gibi, endotel tabakası broşürün yüzeyine kuru steril bir çubukla çevirerek yavaşça çıkarın. Kayma uygulanan dönme yönü ve miktarı numune saflık için kritik öneme sahiptir. Çubukla dönme elinizi doğrusal hareket kontrollü bir kesme oluşturmak için bir ters yönde olmalıdır. Bu kayma, doku ve başka bir şey endotel hücreleri kaldırır ne olduğunu. Kuvvet uygulanan miktar doku direnci hissediyorum, fakat doku içinde nüfuz etmez yeterli olacaktır. </li><li> Bazen, dab kollajenaz çözüm içindeki çubukla ucu lifleri hücreleri çıkarmak için. Sürüntü tamamlandıktan sonra, endotel tabakasının doku öncesine göre biraz daha yumuşak hissediyorum gerekir. </li><li> Hücre süspansiyonu / kollajenaz toplayın ve yeni bir 15 mL steril tüp transferi. Etiket üzerinde yan özgüllük (aynı valf broşürler bir araya toplanmış olabilir) belirtiniz. </li><li> Ventricularis yan adımları tekrarlayın (5,3-5,6) maruz kalan ve böylece yeni bir kültür çanak broşürler aktarın. </li><li> Tüm hücre süspansiyonu / kollajenaz toplandıktan sonra, pelet herhangi bir izole hücreler 5minutes için tüpleri 1000 rpm&#39;de santrifüj ve süpernatant aspirat. Iyi interstisyel hücreler izole Eğer bu hücreler santrifüj edilirken, bu protokol yapmak. </li><li> 3 ml tüpler endotel porcine orta, ikinci kez santrifüj ve medya aspirat. Bu ikinci santrifüj ucu lifleri gibi bazı istenmeyen malzeme filtre yardımcı olur. </li><li> Santrifüje endotel hücreleri, kolaj (santrifüj tüpüne başına 1 balon) ile bir ön kaplı T-25 balon endotel porcine orta ve plaka hücrelerin 5 ml yeniden askıya. </li><li> Hücrelerin endotel orta değiştirmeden önce en az 2-3 gün büyümesine izin. Bu hücreleri kurtarmak ve izolasyon işlemi oldukça sert olduğundan bölmek yardımcı olur. Verim çok düşük olduğunu fark ederseniz, daha küçük bir balon veya iyi plaka hareket, hücre-hücre adezyon, hücre büyümesini teşvik düşünün. Temas inhibisyonu hücre dönüşümün yol açabileceği için izdiham yakın geçiş unutmayın. </li></ol> 6. İnterstisyel Hücreleri İzolasyon <ol><li> Valf başına kollajenaz çözüm (3 broşürler) 10 ml ile 15 ml steril bir santrifüj tüpüne doldurun. </li><li> Endotel hücreleri broşürler sürüntü sonra hemen kollajenaz çözümü ile ilgili 15 mL tüp koyun. </li><li> Yaklaşık 12 ila 18 saat boyunca inkübe edin (hafifçe ajitasyon istenirse). </li><li> Serolojik bir pipet yardımıyla hücre süspansiyonu / kollajenaz homojenize olana kadar bozulmuş doku hafifçe karıştırın. Bu homojenizasyon doku break up ve interstisyel hücreleri serbest yardımcı olur. </li><li> 1000 rpm&#39;de 5 dakika sindirilir doku Santrifüj ve süpernatant aspire. </li><li> 15 mL tüplere 5 ml interstisyel porcine orta, ikinci kez santrifüj ve supernate aspire. </li><li> Santrifüj 5 ml interstisyel porcine orta ve plaka hücrelerin endotel hücreleri yeniden askıya T-75 balonuna (santrifüj tüp başına 1 şişesi kullanın). </li><li> Interstisyel hücre orta değiştirmeden önce en az 1-2 gün hücrelerin büyümesini sağlar. Beklenen endotel hücreleri, fakat çok daha fazla doku enkaz olacak. Hücreler de hücre verim ve doğaları nedeniyle endotel hücreleri daha hızlı izdiham büyümek gerekir. </li></ol> 7. Temsilci Görüntüler <img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Şekil 1" /> Şekil 1: 2-3 gün yazılan izolasyon izole hücrelerinin morfolojisi. (A) VEC tipik bir endotel morfolojisi ve form büyümeyi teşvik etmek için kümeler sergilerler. (B) VIC morfolojisi, genellikle iğsi ve şişeyi boyunca yayılır miyofibroblastlar benzer. <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Şekil 2" /> Şekil 2 confluency izole hücrelerinin morfolojisi. (A) VEC genellikle Arnavut kaldırımlı ve büyüme temas inhibe tipik bir endotel morfolojisi sergilerler. (B) VIC morfoloji, genel olarak iğsi ve büyüme temas inhibe miyofibroblastlar benzer. <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Şekil 3" /> Şekil 3. izole hücrelerinin işlevi özellikleri 6. (A) VEC asetillenmiş LDL alımını yüksek seviyede (kırmızı) ile ilişkilidir. (B) VIC asetillenmiş LDL alımını düşük seviyeleri ile ilişkilidir. <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Şekil 4" /> Şekil 4 izole hücrelerin 6 Hücresel belirteçleri. (A) VEC fenotip Von Willebrand Faktör (yeşil), mavi (çekirdekleri) pozitif boyanma katkıda. (B) VIC fenotip Von Willebrand Faktörü negatif boyama katkıda bulunulmuştur. <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Şekil 5" /> Şekil 5 izole hücrelerin hücresel belirteçleri. (A) alfa SMA (yeşil), mavi (çekirdekleri) için negatif boyama VEC fenotipe katkıda bulunulmuştur. (B) VIC fenotip alfa SMA pozitif boyanma katkıda bulunulmuştur. <table border="1"><tbody><tr><td> Disosiyasyon Ajan </td><td> Disosiyasyon Tekniği </td><td> Hücre Toplama </td><td> Hücre Miktar </td><td> Hücre Saflık </td><td> Bulaşma </td></tr><tr><td> CaCl 2 olmadan EDTA (3mm) </td><td> 5, 20, 60 dk. CaCl 2 ek önce </td><td> 20, 60, 120 dk. toplama önce </td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td> CaCl 2 olmadan EDTA (6mm) </td><td> 5, 20, 60 dk. CaCl 2 ek önce </td><td> 20, 60, 120 dk. toplama önce </td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td> Tripsin-EDTA (0.5 g / L) </td><td> 5, 10, 15 dk. deaktivasyon önce </td><td> Orta hemen toplanan </td><td> + </td><td> + </td><td> + + </td></tr><tr><td> Kollajenaz II (300 U / ml) </td><td> 5, 10, 15 dk. deaktivasyon önce </td><td> Orta hemen toplanan </td><td> -, + + + </td><td> - + + + </td><td> + </td></tr><tr><td> Kollajenaz II (600 U / ml) </td><td> 5, 10, 15 dk. deaktivasyon önce </td><td> Orta hemen toplanan </td><td> + + + + + + </td><td> + + + + - </td><td> - </td></tr></tbody></table> Tablo 1 sonuçtan ön kapak endotel hücre izolasyonu protokolleri.

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Kapak biyoloji bir anlayış izole teknik zorluklar ve kapak endotelyal hücrelerin kültür saf popülasyonları tarafından bozulmuş oldu. Tipik izolasyon teknikleri, altta yatan bazal matris veya endotel yapışkan bağları 2,3 kimyasal disosiasyon enzimatik sindirim içerir . Ön izolasyon deneyleri niteliksel disosiasyon ajanlar ve farklı inkübasyon süreleri değerlendirildi. Bu deneylerin sonuçları, 60 dakika kadar EDTA (veya tripsin-EDTA) kuluçka yerinden oynatmamaya hücreleri başarısız old...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
<tr>
<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
<tr>
<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
<tr>
<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
<tr>
<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
<tr>
<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25384-092</td>
<td>&nbsp;</td>
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<td>60x15 Glass Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-310</td>
<td>&nbsp;</td>
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<td>Paraffin Embedding Wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
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isolation of valvular endothelial cells

Kalp kapakçıkları, kardiyovasküler sistem üzerinden tek yönlü kan akışını sürdürmek için sadece siz sorumlusunuz. Bu ince, lifli dokuların bir ömrü üzerinden birkaç milyar kez açık ve kapalı olarak önemli bir mekanik streslere maruz kalmaktadır. Bu dokuların inanılmaz bir dayanıklılık endotel yerleşik kapak (VEC) ve interstisyel hücreler nedeniyle (VIC), yerel mekanik ve biyolojik sinyallere yanıt olarak sürekli onarım ve bozulmasina. Sadece son zamanlarda, bu hücrelerin in vitro deneyler önemli bir rol oynamıştır eşsiz davranışları anlamak için başladık . Özellikle zorlu VEC izolasyonu ve kültürü. Özel bakım andan itibaren doku son kaplama ile ev sahibi kaldırılır kullanılması gerekir. Burada doğrudan izolasyon, yan özel izolasyon, kültür ve VEC saf popülasyonlarının doğrulanması için protokoller mevcut. Biz nazik bir çubukla kazıma tekniği ile sadece yüzey hücreleri çıkarmak için enzimatik sindirim kullanın. Bu hücreler daha sonra bir tüp içine toplanır ve bir pelet içine santrifüj. Pelet sonra yeniden bekletildi ve kollajen ben matris ile kaplı öncesi kültür şişeler içine kaplama. Büyüme ve PECAM1 (CD31) gibi endotel spesifik belirteçler ifade inhibe VEC fenotip kişi tarafından onaylandıktan sonra, Von Willebrand Faktör (vWF) ve alfa-düz kas aktin (α-SMA) negatif ifade. VEC fonksiyonel özellikleri, yüksek asetillenmiş LDL düzeyleri ile ilişkilidir. Vasküler endotelyal hücrelerin aksine, VEC normalde embriyonik valf 1 oluşumu sırasında meydana gelen mezenşimin haline dönüştürmek için eşsiz bir kapasiteye sahip. Bu aynı zamanda in vitro kültüründe önemli ölçüde uzamış yazılan konfluent sırasında meydana gelebilir, bu yüzden bakım izdiham veya yakınındaki geçit yapılmalıdır. VEC izolasyondan sonra, VIC saf toplumlarda daha sonra kolayca elde edilebilir.

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

Isolation of Valvular Endothelial Cells

Yalıtma ve kalp kapakçığı endotel hücreleri (VEC) saf kültür popülasyonları için bir yöntem sağlar. VEC tüberkülü veya broşürün her iki taraftan da izole edilmiş ve hemen şu olabilir, altta yatan interstisyel hücre (VIC) izolasyonu basittir.

Kapak Endotel Hücreleri İzolasyon

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

Research

JoVE Journal

Biology

20.3K Görüntüleme.  Cornell University. Yalıtma ve kalp kapakçığı endotel hücreleri (VEC) saf kültür popülasyonları için bir yöntem sağlar. VEC tüberkülü veya broşürün her iki taraftan da izole edilmiş ve hemen şu olabilir, altta yatan interstisyel hücre (VIC) izolasyonu basittir.

Video: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

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

İnsan umbilikal ven endotel hücreleri İzolasyon (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biyoloji

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

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

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

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

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

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

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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 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 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 Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Murin Vana Endotel Hücreleri İzolasyon

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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 ...

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 ...