D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

Aksi belirtilmediği sürece tüm işlemler, oda sıcaklığında (~ 23 ° C) gerçekleştirilir. Silikon Master 1. Fabrikasyon <ol><li> O 2 / N 2 gaz altında% 100 etanol veya ksilen ve kuru temiz silikon gofret. </li><li> Bir 0.3-0.5 mikron kalınlığında bir film üretmek için 2000-4000 rpm dönüş hızında spin-lak silikon gofret yerleştirin. </li><li> Bir fotolitografi sistemi kullanılarak Pattern doğru boyutları ile filmin fotorezist </li><li> Tam fotorezist geliştirme çözeltisinin uygun bir hacmi içinde desenli fotorezist kaplanmış silikon gofret bırakın. </li><li> Iyonu giderilmiş su ile geliştirilmiş fotorezist kaplanmış silikon gofret durulayın. </li><li> Yakın dikey yan duvarları, derin reaktif iyon etche bir aşındırma sistemini kullanarak maruz silikon ile alt-mikron ölçeği sırtlar dizileri oluştururlar. </li><li> Bir plazma asher sisteminde silikon gofret yerleştirilerek kalan fotorezist çıkarın. </li><li> W silikon Cutsonraki çoğaltma kalıplama için uygun büyüklükte silikon ustaları içine bir elmas uçlu kesici ile afers. </li></ol> Silikon Master PUA Kalıp 2. Fabrikasyon NOT: nano fabrikasyon için silikon ana eklenecek Volume poliüretan akrilat (PUA) çözeltisi viskozitesi gibi çoğaltılması için nano tel ana alanına bağlı olarak değişecektir. <ol><li> % 100 etanol ya da O 2 / N 2 gaz altında ksilen ve kuru temiz silikon ana yüzey. </li><li> Bir Petri kabındaki silikon ana desen tarafı yukarı bakacak şekilde yerleştirin. </li><li> 2 cm x 2 cm yüzey model, model yüzeyine PUA pipetle 40 ul bir silikon ana için. </li><li> 4 cm tevzi PUA üzerinde x 4 cm saydam polyester (PET) film, bir yaprak yerleştirin. </li><li> Aşağı PET kağıda basın ve böylece (örneğin bir kart gibi) bir rulo veya düz kenarlı yüzey kullanarak desen yüzüne levha altında PUA yayıldı tümmodel PUA ön-polimer ile kaplıdır. </li><li> 50 saniye boyunca yaklaşık olarak 10 cm bir 20 Watt (115 V) UV ışığına (λ = 365 nm) altında silikon ana, Prepolimeri ve PET yerleştirin. Etkili olabilmesi için, UV ışık dalga boyu 310-400 nm arasında herhangi bir yerde olabilir. Işığın yoğunluğu, substratın yüzeyinde 10-15 mW / cm 2 &#39;dir. </li><li> Sertleştikten sonra, forseps ile yavaşça PET filmi çıkarın. PUA silikon ana nanopattern bir negatifi ile PET film eklemek gerekir. </li><li> Kullanımdan önce en az 12 saat boyunca UV altında PUA / PET nanopatterns Cure. Aşırı pozlama bir sorun değildir. </li><li> Silikon ustaları temizlemek için, PUA ilavesi olmadan ana üstüne PET&#39;in başka bir film yer ve 50 saniye boyunca UV ışığı (λ = 365 nm) maruz ve PET filmi çıkarın. Bu, herhangi bir reaksiyona girmemiş monomerler kaldırır. </li><li> O 2 / N 2 gaz altında% 100 etanol veya ksilen ve kuru ile silikon ana durulayın. </li></ol> 3a. Poliüretan polimer Nanopatterning </p&gt; <ol><li> 10 dakika boyunca, bir ozon işleme odası içinde yerleştirilmesi ile 25 mm çapında yuvarlak bir cam slaytlar hazırlayın. </li><li> Kolay kullanım için küçük PDMS blok üzerine ozon tedavi cam slaytlar yerleştirin. </li><li> Cam slaytlar fırça ile yüzeye yapışma yükseltici ince bir tabaka uygulayın. 30 dakika boyunca hava kuru bir cam kayar. </li><li> Yazıcı bir kağıt parçası üzerinde cam slayt yerleştirin. </li><li> Cam slayt merkezine pre-polimer (NOA 76) 10 ul poliüretan (PU) dağıtmak bırakın. Kabarcıklar ek sonra mevcut olduğundan emin olun. </li><li> Sıra PUA kalıp, desen cam slayt üzerine, yüzüstü. Muntazam PUA kalıp boyunca kauçuk bir silindir merdane yuvarlayarak cam slayt yüzeyine karşı PU dağıtılır. Yazıcı kağıt polimer taşma absorbe edecektir. </li><li> Watt UV lamba. PU polimerizasyon zamanı UV kaynağının gücüne bağlıdır. </li><li> UV ışık kaynağından gelen bir numune alın ve dikkatli bir PU kaplı cam slayttan PUA kalıp soyma. PU polimerizasyon c kabul ediliromplete uzak temiz numuneden PUA kalıp peeling ve PU cam slayt bir yanardöner bir görünüme sahip olduğunda. </li><li> Sürece bir ay olarak depolama için desikatörde tamamladı örnekleri yerleştirin. </li></ol> 3b. Nanopatterning Poli (laktid-ko-glikolid) Hidrojel <ol><li> Kuvvetli bir 10:01 oranında silikon elastomer taban ve silikon elastomer sertleştirme ajanı karıştırılarak düz PDMS kalıp oluşturma. </li><li> (PDMS 5 mm kalınlığında olacak şekilde yani) PDMS öncü yemeğin kenarından 5 mm kadar ulaşır, böylece karışık PDMS bir Petri kabı içine çözüm habercisidir dökün. </li><li> Gaz çıkışı için 1 saat boyunca bir kurutucu içinde Petri çanağı ve PDMS öncü yerleştirin. </li><li> Tedavi için en az 2 saat boyunca 65 ° C fırın içine Petri çanağı ve PDMS ön-hareket ettirin. </li><li> PDMS tedavi edildikten sonra, 3 cm x 3 cm kare bölümler halinde düz PDMS kesmek için bir jilet kullanın. Bu desenleme işleminde sonra kullanılan düz PDMS kalıplar olacak. </li><li> Pl tarafından temiz 25 mm çaplı yuvarlak cam slaytlarbir su sonikatöre 30 dakika boyunca, izopropil alkol içinde yüz oluşturma. </li><li> O 2 / N 2 gazı altında kuru temizlenmiş cam slaytlar. </li><li> Damla PLGA solüsyonu 100 ul dağıtılması cam slayt üzerine (% 15 w kloroform içerisinde PLGA v /). </li><li> Çözücü emer ve düz bir PLGA yüzey elde etmek için satış makinası veya ATM PLGA üstüne düz bir PDMS kalıp yerleştirin. 5 dakika boyunca PDMS üstüne bir 200 g ağırlık yerleştirerek bir hafif bir basınç (~ 10 kPa) uygulanır. </li><li> Yavaş yavaş düz PDMS kalıp soyma ve çözgen kalıntılarını uzaklaştırmak ve PLGA ve cam kapak arasındaki yapışmayı geliştirmek için, 5 dakika için önceden ısıtılmış bir plaka (120 ° C) üzerine cam kapak yerleştirin. </li><li> Düz PLGA üstüne NP PUA kalıp yerleştirin ve PUA kalıp üstünde bir 1 kg ağırlık yerleştirerek sabit bir basıncı (~ 100 kPa) ve ısı (120 ° C) 15 dakika boyunca uygulanır ısıtma plakası üzerinde iken. </li><li> PLGA kapak slayt ağırlık kaldırmak ve katmanlarından oda sıcaklığına kadar soğumasını bekleyin. Katmanlarından kanıtlanıncaya kadar PUA kalıp çıkarmayınsoğutuldu. </li><li> Katmanlarından yeterince soğutulduktan sonra, dikkatlice NP PLGA alt katmanı ortaya, NP PUA kalıp soymak. NP PLGA taban bir yanardöner bir görünüme sahip olmalıdır. </li><li> Sürece bir ay olarak depolama için desikatörde tamamladı örnekleri yerleştirin. </li></ol> 4. Hücre Tohum ve Kültür Not: Bu protokol, neonatal sıçan ventriküler miyositlerde (NRVMs) ve H7 insan embriyonik kök hücre türetilmiş kardiyomiyositler (HESC-cm), ancak diğer hücre kaynaklarına kültür açıklar kullanılabilmektedir. <ol><li> 35 mm doku kültürü polistiren çanak ANFS lamelleri (PU veya PLGA) takın. Tabağın tabanına ve yavaşça Norland Optik Yapıştırıcı (NOA83H) Pipet 20 ul NOA üstüne ANFS lamel yerleştirin. Tutkal yaymak ve tüm lamel alt kapağı için izin verin. 10 dakika boyunca UV çanak teşhir ederek NOA Cure. </li><li> Iki kez, 5 dakika boyunca% 70 sulu etanol çözeltisi, 2 ml ile durulanarak ANFS sterilize edin. E çıkarmakaspirasyonu ile etano. İzin ANFS biyolojik güvenlik kabini UV sterilizasyon lambası (λ = 200-290 nm) altında ~ 1 saat boyunca tamamen hava kuru için. </li><li> Hücre yapışma gece boyunca fibronektin içerisinde ANFS kaplanmasıyla geliştirilmiştir. 5 ug / ml DI su içinde fibronektin seyreltin. Çanak içine fibronektin çözeltisi pipetle 2 ml. 37 ° C&#39;de inkübatör yerleştirin ve% 5 CO2, gece boyunca (en az 6 saat). </li><li> Önceki protokollerle 22 göre NRVMs, HESC-CMS, ya da diğer ilgi kalp hücreleri elde. </li><li> Hücreleri peletlemek için 3 dakika boyunca 1000 rpm&#39;de santrifüj hücre örneği. </li><li> Dikkatle aspirasyon ile süpernatant kaldırmak. Pelet rahatsız değil emin olun. </li><li> 4.6 x 10 6 hücre / ml &#39;lik bir konsantrasyona kadar, uygun bir kültür ortamı içinde tekrar süspansiyon hücreleri. </li><li> Dikkatle sterilize ANFS üzerine hücre süspansiyonu 200 ul pipet. Emin hücre süspansiyonu lamel kalır emin olun. </li><li> 37 ° C ve% 5 inkübatörde hücreleri yerleştirinCO hücreleri ANFS takmak için izin 4 saat için 2. </li><li> Aynı koşullar altında kuluçka makinesi içinde hücrelerin çanak ve değiştirmek için sıcak kültür ortamı içinde 2 ml ilave edilir. </li><li> 24 saat sonra ortamını çıkarın ve fazla hücreleri çıkarmak için iki kez 2 ml DPBS ile yıkayın. </li><li> Aynı koşullar altında kuluçka makinesi içinde hücrelerin çanak ve değiştirmek için sıcak kültür ortamı içinde 2 ml ilave edilir. Kültür hücreler çoğalmak üzere. Her gün medya değiştirin. </li></ol>

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Authors have nothing to disclose.

Fonksiyonel olgun kardiyak dokular in vivo ve kardiyak doku mühendisliği in vitro uygulamalarda hem eksiktir. Burada anlatılan CFL Nanofabrikasyona yöntemler hücresel uyumun sağlanması nedeniyle sistemin ölçeklenebilirlik makroskopik doku işlevini etkilemek için güçlü teknikler vardır. Büyük alanlar kolayca desenli ve hücre kültürü için de kullanılabilir. Makroskopik hücresel hizalama biomimetic, o miyokardın 38 mekanik ve elektrik hem özelliklerini etkiler gibi fon...

Kardiyovasküler hastalık dünyada morbidite ve mortalitenin önde gelen nedenidir ve zaten gergin küresel sağlık sistemi, 1,17 üzerinde ağır bir sosyo-ekonomik yükü sunuyoruz. (1) iskemik hastalık veya kardiyomiyopati sonra hasarlı miyokard yeniden veya (2) in vitro ilaç taraması veya hastalık modelleme için kalbin yüksek bir sadakat modeli oluşturmak için: Kardiyak doku mühendisliği iki ayrı hedefleri var. Kalp vücuda kan sağlamak için sürekli çalışmak gerekir karmaşık bir organdır. Kardiyomiyositlerde ve destekleyici dokuların yoğun şekilde paketlenmiş laminar yapılar kalp duvarı 18,19 boyunca sarmal şablonlara göre düzenlenir. Kalp, aynı zamanda etkin bir şekilde elektro gövdesi 21&#39;e kanı çıkarmak için yüksek koordineli bir şekilde 20 bağlanmıştır. Doğanın karmaşık tasarım güvenilir vitro değinmeyecek önce birkaç önemli engeller, ancak, ele alınması gerekmektedir.Sağlam kardiyomyosit farklılaşma yöntemleri 22 geliştirilmeye devam ediyor olmasına rağmen ilk, hPSC-CMs hala oldukça olgunlaşmamış fenotipleri gösterirler. Onların elektromekanik özellikleri ve morfolojisi en yakından fetal seviyeleri 23 maç. Geleneksel kültür koşullarında muhafaza İkincisi, kök hücre-türevi ve primer kardiyomiyositlerde hem doğal, doku benzeri yapılar halinde birleştirmek için başarısız. Bunun yerine hücreler, rasgele yönelimli olmak ve yetişkin miyokard 24&#39;ün bantlı çubuk-şekilli bir görünüm göstermezler. Hücrelerin etkileşim ile hücre dışı matris (ECM) ortamında çok sayıda hücresel süreçleri 11,13,25 önemli bir rol oynamaktadır. ECM önemli hücre 6,26 yapısını ve işlevini etkileyen karmaşık, iyi tanımlanmış moleküler ve topografik ipuçlarının oluşur. Kalp içinde, hücresel hizalama yakından yatan nanometre ölçekli ECM lifleri 2. izler. Bu nanotopograph etkisihücre ve doku işlevini üzerinde iCal ipuçları, ancak, uzak tamamen anlaşılmış değil. Nanometre ölçekli hücre-biyomateryal etkileşimi ön çalışmalar, 27 sinyalizasyon hücre, yapışma 28-30, büyüme, 31 ve farklılaşma 32,33 sub-mikron topografik ipuçlarının potansiyel önemini ve etkisini gösterir. Ancak, tekrarlanabilir ve ölçeklenebilir nanofabricated substratlar gelişmekte zorluk, bu tür çalışmalar, in vivo ECM ortamda kompleksinin çok ölçekli hücresel etkilerini yeniden olamazdı. Bu protokol, doğal kardiyak ECM fiber hizalama taklit eden hücre kültür yapı iskelesi üretmek için basit ve düşük maliyetli bir teknik nanofabrication kardiyomiyosit-biyomalzeme etkileşimlerinin yeni araştırmalar geniş bir yelpazede sağlayan, tarif edilmektedir. Kardiyomiyositler nano ECM çevre ile etkileşim nasıl anlamak daha yakından taklit yerli doku fonksiyon hücresel davranışını kontrol yeteneği için izin verebilirtion. Bundan başka, hücre tekli-tabakaları 3D yapıları ile karşılaştırıldığında basitleştirilmiş bir deneysel sistem ama yine de anlaşılır araştırma ve fonksiyonel tarama 2,34-36 için karmaşık çok-hücresel davranışı sergiler. Son olarak, iskeleler rejeneratif amaçlı 37 için kalp içine implante zaman hücresel greft fonksiyonunu artırmak için kullanılan olabilir.

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			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Name</td>
			<td class="xl63" style="width:66pt" width="88">Company</td>
			<td class="xl63" style="width:66pt" width="88">Catalogue number</td>
			<td class="xl64" style="width:66pt" width="88">Comments (optional)</td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fibronectin</td>
			<td class="xl65" style="width:66pt" width="88">BD Biosciences</td>
			<td class="xl65" style="width:66pt" width="88">354008</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 76</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">7606B</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl65" height="106" style="height:79.5pt;width:66pt" width="88">Surface Adhesion Promotor (Glass Primer)</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">PUA</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MINS-311RM</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Soft Rubber Roller</td>
			<td class="xl65" style="width:66pt" width="88">Speedball</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Silicon Wafers</td>
			<td class="xl65" style="width:66pt" width="88">NOVA Electronic Materials</td>
			<td class="xl65" style="width:66pt" width="88">FA01-9900</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Photoresist</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">SPRT510</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Photoresist Developer</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">MF320</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Electron-Beam Lithography System</td>
			<td class="xl65" style="width:66pt" width="88">JEOL</td>
			<td class="xl65" style="width:66pt" width="88">JBX-9300FS</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Etching System</td>
			<td class="xl65" style="width:66pt" width="88">Surface Technology Systems</td>
			<td class="xl65" style="width:66pt" width="88">NP10 8UJ</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Plasma Asher System</td>
			<td class="xl65" style="width:66pt" width="88">BMR Technology Co.</td>
			<td class="xl65" style="width:66pt" width="88">DSF-200</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Ozone Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-O- 08</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fusion Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-A 11</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 83H</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">8301</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Spin Coater</td>
			<td class="xl65" style="width:66pt" width="88">Laurel Technology</td>
			<td class="xl65" style="width:66pt" width="88">WS-400-6NPP</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Skyrol PET Film</td>
			<td class="xl65" style="width:66pt" width="88">SKC Co., Ltd.</td>
			<td class="xl65" style="width:66pt" width="88">23038-59-9</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">25mm Glass Slides</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">2948</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Sylgard 184 Silicone Elastomer Kit</td>
			<td class="xl65" style="width:66pt" width="88">Dow Corning</td>
			<td class="xl67" style="width:66pt" width="88">6/5/2553</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Poly(D,L-lactide-co-glycolide)</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">P2191-1G</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Chloroform</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">372978-1L</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
			<td class="xl65" style="width:66pt" width="88">Global Insustrial</td>
			<td class="xl65" style="width:66pt" width="88">T9FB503120</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Isopropyl Alcohol</td>
			<td class="xl65" style="width:66pt" width="88">EMD Millipore</td>
			<td class="xl65" style="width:66pt" width="88">PX1835-2</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Hot Plate</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">PC-420D</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Sonicator</td>
			<td class="xl65" style="width:66pt" width="88">Branson</td>
			<td class="xl65" style="width:66pt" width="88">B2510MTH</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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</table>

capillary force lithography for cardiac tissue engineering

Kardiyovasküler hastalık, dünya çapında ölüm 1&#39;in önde gelen nedenidir. Kardiyak doku mühendisliği kalp rejenerasyonu hem de in vitro tarama deneyleri için fonksiyonel doku geliştirme amaçları çığır açan tıbbi keşifler sunmak için çok umut vermektedir. Ancak kalp dokusunun yüksek sadakat modelleri oluşturmak için yeteneği zor kanıtlamıştır. Kalbin hücre dışı matris (ECM) nanometre ölçeğinde 2 mikro-değişen hem de biyokimyasal ve biyomekanik sinyallerin oluşan karmaşık bir yapıdır. Mekanik yükleme koşulları ve hücre-ECM etkileşimleri Yerel son zamanlarda kalp doku mühendisliği 3-5 hayati bileşenleri olarak kabul edilmiştir. Kardiyak ECM büyük bir kısmı bir doku mimarisi ve elektrik kuplajı 2 etkiler nano ölçekli çapları ile uyumlu kolajen liflerinin oluşmaktadır. Ne yazık ki, birkaç yöntem have nanometre ölçeğinde aşağı ECM liflerin organizasyonunu taklit etmek mümkün olmuştur. Nano sentezleme teknikleri son gelişmeler, ancak, kalp 6-9 ECM&#39;nin in vivo yapısal ve alt-tabaka sertliği ipuçları taklit ölçeklenebilir iskelelerinin tasarım ve imalat sağlamıştır. Burada gelişimini mevcut iki tekrar üretilebilir, düşük maliyetli ve biyo uyumlu polimer, poli (laktid-ko-glikolid) (PLGA) 8 ve bir poliüretan (PU) bazlı bir polimer kullanılarak kalp hücrelerinin işlevsel uyum için ölçeklenebilir nanopatterning işlemleri. Bu izotropik nanofabricated katmanlarından (ANFS) iyi organize, hizalanmış dokuların yatan ECM taklit ve hücre morfolojisi ve fonksiyonu 10-14 nanotopography rolünü araştırmak için kullanılabilir. Bir şablon olarak nano tel (NP) silikon ana kullanarak, poliüretanakrilat (PUA) kalıp imal edilir. Bu PUA kalıp daha sonra pa için kullanılırsırasıyla UV-destekli veya solvent-aracılı kılcal kuvvet litografi (CFL), 15,16 yoluyla PU veya PLGA hidrojel ttern. Kısaca, poliüretan ya da PLGA ön-polimer, bir cam lamel ve PUA kalıp üzerine yerleştirilir üzerine uygulanan damladır. UV destekli cfl için, PU sonra tedavi için UV radyasyonu (λ = 250-400 nm) maruz kalır. , Çözücünün aracılık ettiği cfl için, PLGA ısı (120 ° C) ve basınçta (100 kPa) ile kabartılır. Sertleştirme sonrası, PUA kalıp hücre kültürü için bir ANFS geride bırakarak sıyrılır. Bu tür neonatal sıçan ventriküler miyositlerde, hem de insan pluripotent kök hücre-türevi kardiyomiyositlerde Birincil hücreler, ANFS 2 üzerinde korunabilir.

Şekil 1, bu iki üretim yöntemleri için üretim işleminin şematik bir bakıştır. Nedeniyle nano topografya kaynaklanan ışığın kırınımı ile, nanopatterning ANFS yanardöner bir yüzey ile sonuçlanmalıdır. 2 800 nm sırt ve yiv ile iyi desenli 25 mm NP-PU lamel (Şekil 2A) Bu yanardöner yüzey tasvir etmektedir Genişlik (Şekil 2B). ANFS ve yanardöner görünüm sırt ve oluk genişlikleri bağlı olarak biraz deği?...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

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 ...

capillary-force-lithography-for-cardiac-tissue-engineering

Research

JoVE Journal

Bioengineering

12.3K Görüntüleme.  University of Washington. 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.

Video: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Miyokard Doku Mühendisliği Biyolojik Türetilmiş Enjektabl Malzeme imalatı

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Biyomühendislik

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Elastomerik PGS Arter Doku Mühendisliği Yapı iskeleleri

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plazma Litografi Yüzey Desenlendirme Hücre Ağları Oluşturma

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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 ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Doku Mühendisliği için elektrospinning Liflerin Postprodüksiyon İşleme

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Vasküler Doku Rejenerasyon için Mühendislik 3D cellularized Kollajen jeller

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Doku Mühendisliği Thermoresponsive Nanostructured Yüzeylerin Hazırlanması

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Bir ıntrinsic Vaskülarizasyon tarafından Doku Mühendisliği<em&gt; İn Vivo</em&gt; Doku Mühendisliği Odası

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Fare Cochleae iç kulak doku mühendisliği için Decellularizing için bir iletişim kuralı

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...