This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

Bu protokol, hayvan araştırma etik komitesinin LUMC kuralları izler. Araçların 1. Hazırlık, kültür ortamı ve MTCS Not: laminer akış kaputu tüm hazırlıklarını yapın. MTCS perfüzyon odasına, kabarcık kapanı ve tek Lieber ve diğ., 2010, 11 tarif edilmiştir. <ol><li> % 70 etanol ile forseps ve mikro makas dezenfekte edin. Steril Tyrode tamponu (130 mM NaCI, 5.4 mM KCI, 6.0 mM Hepes ve 1.2 mM MgSO 4, 1.2 ile 21G iğne ile 5 ml şırınga hazırlayın mM KH 2 PO 4, 20 mM Glükoz, 1,5 mM CaCl2 .2H 2, O, pH = 7.2). Steril KCI çözeltisi (100 mM) ile, 21G iğne ile 5 ml şırınga hazırlayın. </li><li> 65 kalp başına ortam ml hazırlayın: (A / A, penisilin 100 birim, 100 ug streptomisin ve amfoterisin B 0.25 ug / ml) DMEM,% 10 fetal dana serumu, antibiyotik / antimikotik ile desteklenmiş İnsülin transferinSelenyum (ITS; / ml insülin 10 ug, 5.5 ug / ml Transferin, 6.7 ng / ml sodyum selenit) ve filtre sterilize. </li><li> % 70 etanol ve kağıt havluyla kurulayın sıkarak perfüzyon odası ve kabarcık tuzak sterilize edin. Başlangıçta perfüzyon odasının olmadan, ancak, MTCS (Şekil 1D) birleştirin. Stand kabarcık tuzak yerleştirin. Kolay yükleme pompası başkanları ile bir peristaltik roller pompa kullanın. <ol><li> Silikon boru rezervuar (50 ml tüp) ve pompa, pompa ve kabarcık kapanı, kabarcık kapanı ve rezervuar arasındaki bağlantıları (Şekil 1D bakınız) yapmak kullanma. Orta gaz geçirgen silikon tüp (1 m) ve mümkün olduğunca kısa inkübatör dışına tüp aracılığıyla inkübatör gazıyla gaz dengeye ulaşması için izin yeterince uzun inkübatör içinde boru yapın. </li></ol></li><li> (Düzgün dolaşım sağlamak için / 1 ml civarında dk akış hızı) pompa açmak,% 70 etanol ile 50 ml tüp doldurun ve etanol CIR izinTüm boru sterilize etmek için 30 dakika boyunca hesaplanmalıdır. Rezervuar boşaltma ve sistemden etanol dışarı pompalama yoluyla etanol çıkarın. </li><li> Steril distile su ile 50 ml tüp doldurun pompa açmak ve 30 dakika boyunca su sirküle etanol kalan kurtulmak için izin verin. Rezervuar boşaltma ve sistemden suyu pompalanarak suyunu çıkartın. </li><li> Orta 45 ml, 50 ml tüp doldurun, inkübatör standı koymak. Pompa (1 ml / dakika akış hızı yaklaşık) açın ve tüm boruların doldurmak için orta sirküle, tüm hava kabarcıklarını çıkarmak ve orta en az 1 saat boyunca kuluçka makinesi içinde, gaz bileşimine uyum sağlar. Standart doku kültürü koşullarında (% 5 CO2 ve 37 ° C) kuluçka tutun. Hiçbir kabarcıklar tüp mevcut olduğundan emin olun. </li></ol> Fare Heart 2. izolasyonu <ol><li> Heparin 500 adet fare ile enjekte edilir. Bu pıhtılaşma kan önlemek ve b kaldırılmasını sağlar olacakkalp LOOD. 10 dakika sonra, hassas bir buharlaştırıcı kullanılarak% 4 izofluran ile bir indüksiyon odasında fare anestezi. Fare ayak tutam tepkisiz olduğunda anestezi yeterlidir. </li><li> Bir diseksiyon kurulu fareyi aktarın ve koaksiyel devresine bağlı bir yüz maskesi vasıtasıyla anestezi korumak. % 70 alkol ile fare kürk dezenfekte edin. Makas karın boşluğuna açılması kullanma vena kava ve diyaframı ortaya çıkarmak. </li><li> , Kalp maruz geçen ilk kaburga başlayan yanal kesiler yapmak ve farenin başının üzerinde göğüs kafesi yansıtacak. Fare artık nefes almıyorsa olarak anestezi durdurun. Kalp atışını gözlemleyin. </li><li> (Diyafram üzerinden) göğüs boşluğuna karın gelen inferior vena kava içine steril Tyrode Tamponu içeren 5 ml şırınga bağlı 21G iğne takın. Kan iğne yerleştirilmesi gelen vena kava kaudal çıkabilirsiniz emin olun. Inci serpmeke Tyrode Tampon nazikçe ve vena kava içine sabit basınç kalp kısmen kırmızı rengini kaybeder kadar. Not: Kalp kalpten kanın çıkarılmasını sağlayan yenerek kalır. </li><li> Steril KCI çözeltisi ihtiva eden bir 5 ml şırıngaya bağlanmış 21G iğne ile iğne değiştirin. Yavaşça kalp atışını durana kadar vena kava içine KCl çözeltisi serpmek ve iğneyi çıkartın. Not: KCl çözümü koroner sistemin daha kolay perfüzyon iğne yerleştirilmesi ve perfüzyon sağlayacak rahat diyastolik aşamasında kalbi korur. </li><li> Hafif kavisli bir forseps kullanarak kalbi kaldırın ve makas kullanarak kalp sağlam ve kalbe bağlı proksimal arter ve venler çevreleyen dokudan serbest kalbini incelemek ama bırakın. Buz soğukluğunda PBS antibiyotik / antimikotik (A / A) ile desteklenmiş ihtiva eden bir 15 ml tüp kalp aktarın. Kadar 3 saat boyunca buz soğukluğunda PBS kalp saklayın. </li></ol> 3. CannulatioPerfüzyon Odası Fare Kalpler n <ol><li> Laminer akış kaputu tüm prosedürleri uygulayın. 10 cm&#39;lik Petri kabı 15 ml tüp izole kalbi aktarın ve eklemek PBS A / A ile desteklenmiş. </li><li> Bir mikroskop altında, tüm non-kardiyak doku kaldırmak için mikro makas ve forseps kullanabilir ancak artan aorta korumak kalbe brakiyosefalik arter ve pulmoner venlerin, 2 mm proksimal en az çatallanma için. Sol ventrikül lümen (tipik olarak 2 mm) erişim temin etmek kalbin üst ucu kesin. Diseksiyon mikroskobu altında akış kaputu perfüzyon odasına yerleştirin. </li><li> Silikon tüp kullanılarak uç iğneye orta ile 5 ml şırınga takın. 20 ml ortam ile perfüzyon odasına doldurun ve perfüzyon odasında 2 körelmiş iğneler (Şekil 1) arasındaki dönme sahneye kalbini koydu. Kalp önüne konumlandırılmış böylece dönme aşamasında yüksekliğini ayarlayıniğne. </li></ol> Mitral Vana Kültürleme 4. ligasyonu (Şekil 1) <ol><li> Sütür (ipek 7.0) ile aort Arter. Sütür ile sol atriyal apendiks Arter. Sol atriyuma pulmoner ven yoluyla (Şekil 1&#39;de. 1 nr) iğne takın ve sol atrium onun giriş sütür proksimal ile Arter. Bu mitral kapak zedeleyecek şekilde iğne sol atrium içine çok uzak değil emin olun. </li><li> Sol ventrikül içine doğrusal hareket ile iğne (Şekil 1 &#39;de No.2) yerleştirin. 50 ml&#39;lik bir tüpe perfüzyon odasından gelen orta aktarın. Biyouyumlu yapıştırıcı ile miyokardiyuma iğne Seal. </li><li> Tutkal kuruduktan sonra dikkatlice nr.1 iğne ve herhangi bir sızıntı olup olmadığını kontrol etmek için bir orta dolu şırıngayı bağlayarak kalp içine orta enjekte. İğne Nr.2 ve son kalan kan kalbin dışına perfüze o orta çıkar olun. </li></ol> 5. LiAort Vana Kültürleme için Navigasyon (Şekil 1) <ol><li> Aort iğne (Şekil 1 &#39;de No.1) takın ve sütür ile ligate. Bu aort kapağını zedeleyecek şekilde iğne aort içine çok uzak değil emin olun. Sol ventrikül içine doğrusal hareket ile iğne (Şekil 1 &#39;de No.2) yerleştirin. 50 ml&#39;lik bir tüpe perfüzyon odasından gelen orta aktarın. Biyouyumlu yapıştırıcı ile miyokardiyuma iğne Seal. </li><li> Tutkal kuruduktan sonra dikkatlice Nr.2 iğne ve herhangi bir sızıntı olup olmadığını kontrol etmek için bir orta dolu şırıngayı bağlayarak kalp içine orta enjekte. İğne nr.1 ve son kalan kan kalbin dışına perfüze o orta çıkar olun. </li></ol> Stand 6. Perfüzyon Odası <ol><li> Orta aktarıldı 20 ml perfüzyon odasına kadar doldurun. Odasının üzerine conta ve kapak yerleştirin ve pul ve vida ile sıkın. </li><li> Incuba monte MTCS kaldırtor. Stand perfüzyon odasına takın. Boru bağlayın (Şekil 1D bakınız). Inkübatör (% 5 CO2 ve 37 ° C) &#39;de standı yerleştirin. Pompanın hortumu bağlayın. 600 ul / dk etrafında akış hızı ile pompa açın. </li><li> Rezervuar ve / veya kabarcık kapanı orta düzey gelen orta damlaların düşerek akışının varlığı görselleştirme izin verdiğinden emin olun. Kültürden sonra, kalp, sistemden çıkarılır fikse edilmiş ve histolojik inceleme için işlenir. </li></ol>

<ol>
	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

Kalp fare vanaları kültürleme Kritik adımlar akış yönünü doğru sağlamak için fare kalbin eksizyonu ve vanalar dik iğne canlılığını ve ligasyonu sağlamak mümkün olduğunca kısa perfüzyon odasında ligasyon arasındaki süre yapım dahil . Ayrıca, orta olmadan perfüzyon odasında bağlanmasından sonra akışını kontrol uygun ekleme ve iğneler bağlanmasını sağlar. Bu steril kültür korumak ve potansiyel akış tıkayan kalbinde sıkışıp alabilir boru, hava kabarcıklarını engellemek i...

Kalp kapak hastalığı, Batı dünyasında morbidite ve mortalitenin önemli bir nedenidir; prevalansı yaşla birlikte artar ve nüfus 75 yıldan fazla% 10 ve 1 yaşlı etkiler. Kalbin sistemik kısmının vanalar, aort ve mitral vanalar, en çok etkilenen bulunmaktadır. Kalp kapakçığı hastalığı mekanik özellikleri 2 değişmesi ile sonuçlanır valf yüksek organize yapı kaybıyla karakterizedir. Yapısal bütünlük valfi işlevi açısından kritiktir. Vana bildiriler kapak interstitial hücrelerin (VIC), kapak endotel hücreleri (VEC) ve yüksek bir tabakalı desen 3,4 düzenlenen hücre dışı matris oluşur. Kurbanlar ECM sentezi, yıkımı ve organizasyon sorumludur. Faktörler kan dolaşımına, EC kaynaklanan veya fonksiyonlarını düzenlediğini kurban ECM hareket ikamet eden. Ek olarak,mekanik kuvvetler Laminer veya salınımlı kayma gerilmesi, kurban 5 davranışını etkileyen basınç veya çekme gerilmeleri sonucu kardiyak döngüsü sırasında broşür hareket. Vananın yapısı nasıl düzenlendiği anlamak için, öncelikle kurban kardiyak döngüsü sırasında yaşanan uyaranların farklı setine nasıl yanıt anlaşılması gerekir. In vitro çalışmalar, kapak hücrelerinin özellikleri ve yetenekleri hakkında çok bilgilendirici olmuştur. Bu hücrelerin in vitro yanıtı, ancak, her zaman doğru bir in vivo yanıt 6 taklit olmayabilir; örneğin, uyaranlara VIC tepki EC varlığı ve ECM bileşimin 5 bağlıdır. Ayrıca, uyaranlara kapak hücrelerinin tepki broşürü 7 onların belirli bir konuma bağlıdır. Biyokimyasal uyaranlara ek olarak, kapak hücrelerinin davranışı o hareket mekanik kuvvetlerin belirlenirn valfi 8. Vananın Her bölgenin hemodinamik gerilmelerin kendine özgü kümesine tabi tutulur. Geçerli ex vivo modeller mekanik kuvvetler kapak yapısının 5 önemli belirleyicileri olduğunu göstermiştir rağmen, ilgili mekanizmalar hala belirsiz. In vivo modeller kapak gelişimini 9,10 altında yatan moleküler mekanizmaları içgörü sağladı ederken, yetişkin kapak biyoloji içine anlayışlar hala zor olduğunu. Bu nedenle, bir ex vivo akış modeli olan kalp valfleri zaman 11 arasında bir süre için merkezinde kendi doğal pozisyonunda kültürlenebilir geliştirilmiştir. Bu valfler doğal konfigürasyonda kalır ve kurban mümkün olduğunca doğal uyaranlara karşı yanıt kurban yapım in vivo aynı ortamı deneyim avantajına sahiptir. Buna ek olarak, kalbinde doğal pozisyonda vanaların kültürü her tabi kolaylaştırırİlgili hemodinamik gerilimlere kapak bölgesi. Örneğin, bu ex vivo modelde, Minyatür Doku Kültür Sistemi (MTCS), valfler kalp kapağı yeniden rolleri incelenmesi sağlayan farklı biyokimyasal ve hemodinamik uyaranlara maruz bırakılabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Aort kapak (Şekil 2) ya da mitral kapak 11, en ​​az 3 gün süre ile kültüre edilebilir. (Aort kapağı ve mitral kapağın diyastolik pozisyon için sistolik konumu temsil) açık konumdayken kültürleme yoluyla, kapak hücreleri canlı kalır. TÜNEL-pozitif hücreleri (Şekil 2H, I) veya ayrıldı kaspaz-3 yokluğu ile belirlendiği gibi Resim hücre ölümü gözlenir (gösterilmemiştir olup Lieber ve diğ., 2010 11). (Masson ...

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Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Minyatür Doku Kültürü Sistemde Fare Kalp Vanalar Kültürleme

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.7K Görüntüleme.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Video: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Proteolitik Degradable 3 Boyutlu Sistem Yumurtalık Folikül Encapsulation ve Kültür Bir Yöntem

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Biyomühendislik

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Kalp Doku Mühendisliği Fibrin Hidrojel kardiyomiyositlerinin Kapsülleme

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kardiyak Doku Mühendisliği kılcal Kuvvetleri Litografi

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

Kontrast-bilgisayarlı Perfüzyon-sabit Örneklerinin Tomografi kullanarak İnsan Kardiyak Venöz Sistemi anatomik yeniden düzenlemesi

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Tri-broşür Polimer Vanalar göreceli hidrodinamik Değerlendirme Protokolü

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

İnsan 3D Doku Mühendisliği<em&gt; In vitro</em&gt; Tümör Test Sistemi

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

bir<em&gt; İn Vitro</emKemirgen İntervertebral Disk&gt; Organ Kültürü Modeli

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Üç boyutlu peptid iskele memeli hücrelerinde kültür

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Net kalıp tabanlı yöntemi iskele ücretsiz üç boyutlu kalp doku oluşturma

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Atriyoventriküler kalp kapakları biaxial mekanik karakterizasyonu

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...