The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.

Tüm fare deneyleri ABD Hayvan Refahı Yasası uyarınca yapılmış ve Minnesota Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. Fare Kalp İzolasyon 1. Yöntem <ol><li> tek kullanımlık bıçak ile kafaları kesilmek suretiyle bir yenidoğan fare Euthanize. </li><li> % 70 etanol ile toraks çubukla. </li><li> # 5 forseps bir çift ile yanal cildi çekerken standart makas ile göğüs duvarından onu uzak keserek göğüs cildi parçalara ayır. </li><li> Karın duvarından keserek makas ile sternum sadece alt karın Perfore. makasla göğüs iki tarafında kaburga olsa kesim sırasında 5. forseps ile kılıç şeklinde sürecini kavratmak, vücuttan rostrally sternum geri çekin. göğüs kafesi kalbi ortaya çıkarmak için forseps ile superior yansıtılır. </li><li> Açık açık teşhir, 5. forseps ile yanal çekerek timus iki ana lob teşrihAort kemer yanı sıra kaval ve pulmoner venler. </li><li> 10 cm yaylı makas ile aortik arktan önemli arterler, aorta kendisi transect. Kalbin taban ve brakiyosefalik arter arasında aort saklayın. trakea ve özefagus ayıran, öne kalbi yansıtmak için # ile 5 forseps kopmuş gemilerin uçlarını kavrayın. </li><li> 10 cm yaylı makas ile, kalbin her iki tarafta da akciğer ve kalp arasında keserek, pulmoner damar ve diğer büyük damarlar transeksiyonu. damarlar drenaj sağlamak için açık kalır. </li><li> büyük damarların kesik kenarlarından tutarak, 5. forseps ile mediastinumdan kalbi çıkarın. kateterizasyon için steril fosfat tamponlu tuz (PBS) ihtiva eden bir 60 mm kültür çanağı kalbi yerleştirin. </li></ol> Langendorff perfüzyonu ile Decellularization 2. Yöntem <ol><li> önceden kateter hazırlayınız. 4 cm&#39;lik bölümünü çizmekPE küçük bir alkol alevinde 50 boru küçük, daha ince kateteri oluşturun. Boyutları karşılamak için bir bıçak ile biter süs (Yaklaşık. Yakl. 500 mikron OD bir flanş ile 300 mikron dış çap OD) Şekil 1&#39;de gösterildiği. Bu çekme her iki tarafında iki simetrik kateterleri sağlayacaktır. </li><li> İnce ucunda açılış üzerine flanş eritmek için, kısaca ateşe hortumun kesik ucunu yönlendirin. </li><li> PBS ile 12 ml şırınga doldurun ve şırınga 3-yollu stopcock koyarak kateter monte edin. stopcock bir 22 G x 1 iğne ekleyin ve septum boyunca iğne sürücü. Iğne (Şek. 1) üzerine çizilmiş PE boru kateteri kaydırın. </li><li> tüm hava kabarcıkları bertaraf edildiğini güvence PBS ile monte parçaları sulamak. </li><li> aort kapak geçmiş uzanan değil, izole kalp aort içine, yukarıda özetlenen ve 7-0 sütür bir kravat Arter olarak hazırlanan kateteri, yerleştirin. t flanşı istirahatO sıkı bir mühür yapmak ve kateter çıkmasını kalbi önlenmesi, kravat karşı proksimal kateter. </li><li> nazikçe PBS içeren şırınga kullanarak kalbi perfüze iken, büyütme altında kateterizasyon gözlemleyin. Orada hiçbir sızıntı sistemde ve gizli kan kaldırılır gibi doku homojen blanches emin olun. </li><li> giriş adaptörü boynuna septum yerleştirin ve cam üzerinde tarafı katlanarak mühür. </li><li> Şekil l&#39;de gösterildiği gibi, 20 mm Hg basıncı üreten bir sütun üretilmesi için yeterli uzunlukta bir hat ile distile su içinde% 1 sodyum dodesil sülfat (SDS), 60 ml (DH 2 O) ile dolu hazne takın. 1. 1 mm Hg ilişkisine dayalı sıvı sütunu yüksekliği baskısı hesaplayın 1,3595 cm H 2 O eşittir Dikkat: SDS tahriş edici özelliklere sahip son derece yün tozdur. İletişim kaçınılmalıdır. Uygun koruyucu giysi kullanılarak toz anlaştım. </li><li> 14 saat boyunca,% 1 SDS ile kalp serpmek. Kalp gözlemlenebilir kalan doku ile görünüş olarak saydam olacak. </li><li> Kalan SDS çözeltisi musluk sistemi kadar yıkayın ve 10 ml dH 2 O ile değiştirin 1x penisilin streptomisin (LTR yükseltici) durumunda ihtiva eden 60 ml PBS ve ardından 10 mi dH 2 O ve ardından (damıtılmış su içinde seyreltilmiş), 10 ml% 1 Triton X-100 ile kalp, serpmek. </li><li> 4 ° C&#39;de 1x Pen-Strep ile PBS içinde kalp saklayın. amaçlanan sonrası decellularization uygulama daha sonra aorta kateter sağlanmalıdır perfüzyon gerektiriyorsa, aksi takdirde dikiş kravat kesmek ve kateter çıkarın. </li></ol> 3. DNA tayini <ol><li> 50 mM KCI 200 ug / ml proteinaz K ile hücresizleştirilmiş ECM veya bir kalp atım bir özetini Hazırlama, 2.5 mM MgCI, 10 mM Tris (pH 8.3) içinde bir 2,% 0.45 Tween 20. </li><li> Doku çözünene kadar çalkalanarak 55 ° C&#39;de doku inkübe,4-5 saat. </li><li> Tahlili 4,11 bağlanma DNA ile homojenat DNA içeriği nicelleştirmektedir. </li></ol> 4. Sabitleme ve Doku Kesit <ol><li> Oda sıcaklığında 30 dakika boyunca PBS içinde% 4 paraformaldehid hücresizleştirilmiş, recellularized veya kontrol P3 kalpleri düzeltildi. </li><li> PBS 3x doku yıkayın. </li><li> batar kadar 4 ° C &#39;de 0.1 M fosfat tamponu içinde% 7.5 sükroz, bir solüsyon içerisinde bulunan dokunun yerleştirin. </li><li> batar kadar, yine 4 ° C &#39;de 0.1 M fosfat tamponu içinde% 15 sakroz çözüm değiştirin. </li><li> 37 ° C doku, sıcak% 15 sakaroz ve 0.1 M fosfat tampon maddesi içinde bir jelatin çözeltisi ile hacminin yarısı yerine ve bir gece boyunca dengelenmeye. jelatin konsantrasyonu% 1,% 2.5,% 5 ile adım adım artan ve son olarak% 7.5 iki kat. </li><li> Taze% 7.5 jelatin kullanan cryomolds örnekleri yerleştirin. decellularized numunelerin şeklini geri, sıvı jelatin t olarak odalarına perfüze edilebilirO kalpler kalıplanmıştır. sıvı azot üzerinde cryomolds yüzen ile dondurun. -80 ° C&#39;de saklayın. </li><li> Kriyostat ile kesilir (10 mikron kalınlığında) kesitler. bölüm yüzeyine yakın slayt getirin ve bölüm nedeniyle elektrostatik tedavi slaytlar üzerinde yüke yüzeye bıçak kapalı hareket gözlemlemek. bir gecede slaytlar kurulayın ve gelecek analizi için -80 ° C&#39;de saklayın. </li><li> dondurucu Slayt çıkarmak Oda sıcaklığına ve daha sonra jelatin çözmek için 37 ° C &#39;de 20 dakika süre ile PBS içeren bir Coplin kavanoza yerleştirin. </li><li> Hematoksilen ve Eosin ile Adım 4.7 kesilen bölümleri Leke. Bir Coplin kavanoza yerleştirin kayar. 1.5 dakika boyunca maddesi mavileştirici 1 dakika boyunca musluk suyu, 3 dakika boyunca tampon, 1.5 dakika boyunca musluk suyu, 45 sn için hematoksilen Adım 4.8&#39;de sulu slaytlar Açığa,% 80 etanol, 1 dakika boyunca, Alkol Eosin Y 8 saniye, 1 dk 3x 1 dakikada, 1 dakika 2x% 100 etanol maddesi temizleme (ksilen yerine)% 80 etanol, reçine ile lamelgöre montaj ortamı. mikroskopik slaytlar inceleyin. </li><li> nemli bir oda içinde hidratlanmış slaytlar koyarak bu kolajen IV, ECM yapısal proteinler kalp ECM, leke ECM proteininde reaktivite tutma teyit ve% 0.1 Triton ile fosfat tamponlu tuzlu su içinde% 10 normal eşek serumu (NDS) ile tedavi edilmesi için -X100 oda sıcaklığında 1 saat (RT) (PBST). Doku bölümleri kapsayacak şekilde yeterli hacme kullanın. </li><li> % 5 NDS / PBST bir ampirik olarak tespit seyreltme de tercih birincil antikor ile çözüm değiştirin. Örneğin, kolajen IV antikoru 1 ile seyreltildi: 150. 4 ° C&#39;de bir gece boyunca inkübe edilir. </li><li> PBST 3x ile slaytlar yıkayın ve bir floresan boya ile konjuge seçim ikincil antikor uygulayın. bir ampirik olarak tespit miktarda antikor sulandırmak. Oda sıcaklığında 1 saat süreyle inkübe edin. PBS 3x yıkayın. </li><li> DAPI DAPI içeren mounti kullanılarak hücre çekirdeğinin olmaması doğrulamak için gibi bir DNA bağlayıcı boya ile slaytlar LekeKapak slaytlar kayma sırasında orta ng. </li><li> 400X&#39;lik 50 bir floresan mikroskop ile slaytlar inceleyin. </li></ol> Recellularization 5. P1 Neonatal Fare Ventriküler Kardiyomiyositler <ol><li> % 70 etanol ile her yavru Sprey ve tek kullanımlık bıçak ile başını kesmek. </li><li> toraks göğüs boşluğu maruz küçük steril makas ile orta hatta bölünmüş ise başparmak ve işaret parmağı arasındaki her yavru tutun. o büyük damarların ve sadece ventriküler doku bırakarak kulakçıklar serbest kesilir iken kalp göğüs serbestçe çıkıntı izin basınç uygulayın. </li><li> 20 ml buz gibi soğuk Kalsiyum, bütün kalpler toplanana kadar, 20 mM 4- (2-hidroksietil) -1-piperazinetansülfonik asit (CBFHH) sahip bikarbonat bilgilerini Hank tamponlu tuzlu su çözeltisi içeren 50 ml&#39;lik bir tüp içine doğrudan her kalp yerleştirin. </li><li> CBFHH aspire ve boru taze CBFHH (buz soğukluğunda) 10-15 ml ekleyin. t doku dönen dokuyu yıkayıno tüp birkaç kez. </li><li> 60 mm plastik Petri kabı içine kalpleri içeren solüsyon süzün. </li><li> uzakta yaylı makas ile kalpleri onu kırparak buz üzerinde büyütme altında kalpleri herhangi gizli atriyal doku teşrih. homojen büyüklükte küçük parçalar halinde ventriküller kadar kıyma. 5. forseps ile çanak herhangi ayrılmış olmayan ventriküler doku çıkarın. </li><li> steril mikro karıştırma çubuğu içeren küçük steril şişenin içine doku parçaları aktarmak için gerekli olduğu gibi CBFHH çözümünün kadar Pipet. </li><li> Doku flakon dibine tortu ve CBFHH çözüm kaldırmak için izin verin. </li><li> CBFHH bir enzim çözeltisi 1.75 mg / ml tripsin, 20 ug / ml DNaz II 50 ml hazırlayın. Pipet 5 Bu enzim solüsyonu ve bir manyetik karıştırıcı ile kıyılmış ventriküler doku ve yer ihtiva eden bir tübe ekleyin. 8 dakika boyunca, oda sıcaklığında sabit bir oranda (340 rpm) karıştırılır. NOT: karıştırma eylem nazik olun ve henüz izin vermelidirbulamaç durdurulması için. </li><li> karıştırma plakasından şişe çıkarın ve bulamaç 3 dakika tortu izin verir. öncelikle kan hücreleri ve doku artıkları içerdiğinden pipetleyin, ilk sindirmek süpernatant atın ve. </li><li> Pipetleyin ikinci 5 ml enzim çözeltisi kısım ve sindirerek ventrikül ekleyin. 8 dakika boyunca karıştırın ve bir diğer 3 dakika için tortu sağlar. Önceki digests başlamadan, iki adet 50 ml tüpler (1 ve 2 ile) hazırlanması Her biri 12 ml buz gibi soğuk fetal sığır serumu (FBS). Her bir tüp üzerinde bir 40 mikron hücre süzgeç yerleştirin. Pipet tüp 1 filtre aracılığıyla bu yüzer. </li><li> FBS içeren iki tüp arasında sindirmek süpernatant yerleştirilmesini alternatif, sindirim sürecini bir ek 8 kez tekrarlayın. Her bir tüp içinde eşit hacimlerde sağlamak için tüpler 1 ve 2 arasında eşit Son süpernatant bölün. NOT: Her toplama tüpü şimdi hücre süspansiyonu 32.5 ml toplam hacmi içerecektir. </li><li> tüpler içerirler santrifüj4 ° C&#39;de 6 dakika boyunca 150 x g&#39;de süpernatan ing. </li><li> Sindirim alikotları toplarken, plaka başına (% 10 FBS, 1 x Pen-Strep, 1 x L-glutamin ile Dulbecco Modifiye Kartal Ortamı (DMEM)) ortam 6 ml beş 100 mm doku kültürü plakaları hazırlamak. Ortamı dengelenmeye 30 dakika için 37 ° C,% 5 CO2 seviyesinde bu alikotları inkübe edin. </li><li> Her iki toplama tüpleri hücreleri birleştiren CBFHH enzim karışımı aspire ve yukarıda hazırlanan kültür ortamı 30 ml tekrar süspansiyon hücreleri. </li><li> 100 mm levhaların her hücre süspansiyonu 6 ml dönün ve 37 ° C&#39;de 45 dakika inkübe edilir. </li><li> (Fibroblast fraksiyon yemekler bağlı başlamış ve eğer istenirse ayrı ayrı yetiştirilebilir) inkübasyonun sonunda, 5 yeni yemeklere yıkanarak yapışmayan hücreler transferi ve 45 dakika boyunca tekrar inkübe edin. </li><li> Ön kaplama ikinci tur sonunda, yemekler medya ve yapışmaz hücreleri toplamak ve 150 ° C&#39;de medya dönmeye6 dakika xg. </li><li> (Perfüzat 100 ul Yapı başına 4.0 x 10 5 hücre), yaklaşık istenen konsantrasyona hücreleri getirmek için fazla ortamı dışarı çekilmesi. Sayımı ve veya canlılık testi 14 için bir numuneyi çıkarın. </li></ol> P3 Kalp Matrix 6. Biyoreaktör Recellularization <ol><li> bir gecede hücrelerin eklemeden önce kültür ortamı (Adım 5.15 bakınız) ile izole kalp matrisi ön işleme tabi. </li><li> Şekil l&#39;de gösterildiği gibi, bir Langendorff sistemi elemanları bir araya getirin. 2. Cam parçaları Otoklav ve etilen oksit sterilite sağlamak için plastik parçaları sterilize edin. sistemin çeşitli alt birimleri destek standına monte edilmeden önce, bir laminar akış başlığı içinde monte edilebilir. Dolaşan su banyosu ve ısıtılmış su akış yolu açıklık amacıyla çıkarılmıştır. </li><li> doku kültür ortamı 120 ml monte sistemi doldurun (Adım 5.15 bakınız). </li><li> 60 mm cul Kademe 2.14 den kateterize kalp matrisi yerleştirinbüyütme altında ture bulaşık kateter Adım 5.20 hücre süspansiyonu ile yüklü x 1 iğne 22 g ile 1 ml şırınga bağlanıp. Bu montaj kalbi embolizan önlemek için hava kabarcıkları serbest olduğundan emin olun. </li><li> Yavaşça koroner arterler ile kalp matriks içine hücre süspansiyonu 100 ul serpmek. Tamamlandığında, şırınga / iğne kombinasyonu ayırın. NOT: Yaklaşık bir yavaş perfüzyon. min başına 20 ul kalbi transit, damarların dışarı akan hücreleri önlemek için gereklidir. </li><li> biyoreaktör kalp kavanoz kapağının alt kısmında Luer bağlantısına sabitlenmiş bir 22g künt saplama, saplama üzerine kateter itin. </li><li> Peristaltik pompayı Şekil 2&#39;de belirtildiği gibi işlemin akışı gözlemleyin. Hazneden işlemin akışı Aşama 2.5 aort yerleştirilen kateter (Şek. 2 kırmızı yolu) kabarcık kapanı vasıtasıyla pompa yoluyla. damar dışına kalp dolaşım akışını gözlemlemekMedya rezervuar apeks, çevrimli gelen s ve damla. Not: perfüzyon akış hızı, yapısı damar direnci ve kalp boyutu ancak 100 ul 50 oranında bireysel etkenlere bağlıdır / dakika için iyi bir başlangıç ​​noktasıdır. ara zaman noktalarında, fonksiyonel değerlendirmeler yürütülebilir. Neonatal recellularized kalp boyutu ölçeği bu gerçekleştirilebilir ama optik tabanlı sistemler yetişkin fare kalplerinde kullanılmıştır gibi davranış yenerek ölçmek için kullanılabileceğini belirledik, değerlendirme sınırlar. 4 yapı uzatılmış için perfüze edilebilir (23 saat kadar) süreler. </li></ol>

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Y., 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=Repopulation+of+decellularized+mouse+heart+with+human+induced+pluripotent+stem+cell-derived+cardiovascular+progenitor+cells.">Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells.</a> Nat Commun. 4 (2307), 1-11 (2013).</li></ol>

The authors have nothing to disclose.

Kalbin tekrarlanan perfüzyonları bu tekniğin bağımlılığı bir emboli kaçınma başarılı bir sonuç önemli bir bileşeni yapar. Adımlar 2,8-2,14 arasında çözümün değişikliklere Adımlar 2,2-2,6 kalbin ilk kateterizasyon itibaren, hangi taviz miyokard içine perfüzat akışını hava kabarcıklarının giriş izin verebilir manipülasyonlar vardır. Nedeniyle yenidoğan kalp küçücük boyutuna, damarsal bile dakikalık kabarcıklar böylece eksik decellularization render, bir teknik infarkt neden ol...

Kalp yetmezliği yaygın ve ölümcül olduğunu. Bu organlara kan akışını bozar ve vücut karşılanmamış metabolik taleplerini bırakır kalp, azalmış kontraktilite ile sonuçlanan ilerleyici bir hastalıktır. 5,7 milyon Amerikalı kalp yetmezliği olduğu tahmin ve Amerika Birleşik Devletleri&#39;nde 9 hastanede birincil nedenidir edilir. Amerika Birleşik Devletleri&#39;nde kalp yetersizliğinde hastaların tedavisinde kolektif maliyet yıllık 9-10 başına 300 $ milyar doları aşmaktadır. son dönem kalp yetmezliği için tek kesin tedavi ortotopik kalp naklidir. Her yıl, 100.000&#39;den fazla verici kalplerinde Birleşik Devletler 1-2&#39;de kalp nakli işlemleri için gerekli olduğu tahmin edilmektedir. Nedeniyle donörlerin sınırlı sayıda için, sadece yaklaşık 2.400 nakli ABD 2 her yıl yapılmaktadır. Açıkçası, bu organ yetersizliği diğer stratejiler nakline için ek organları üretmek için gerekli olarak ele alınması gerekenreddi ve kullanım süresi bağışıklık bastırılması ile ilişkilidir komplikasyonları önlemek için yon ve ideal olarak, bu organlar otolog olabilir. Memeli yetişkin kardiyomiyositlerde yaralanması üzerine sınırlı bir rejeneratif kapasitesini göstermek ama son kanıtlar memeli yenidoğan kalpleri yaralanma 5-8 aşağıdaki olağanüstü rejeneratif kapasitesini korumak olduğunu göstermektedir. Özellikle, kısmi cerrahi rezeksiyon sonrasında bir rejeneratif pencere doğum ve doğum sonrası gün arasında bulunmuştur 7. Bu rejeneratif dönemi fibrotik skar eksikliği, neovaskülarizasyon oluşumu, epicardium gelen anjiyogenik faktörlerin salınımı, kardiyomyosit çoğalması 5-8 ile karakterizedir 11. Bu zaman reaktif pencere biyoyapay kalbin gelişiminde malzemenin yeni bir kaynak olarak yenidoğan kalp kullanarak potansiyel sağlar. hücre dışı matris cardiomyocyt teşvik etmek için önemli ipuçları sağlamak için bilinene çoğalması ve büyümesi. Yenidoğan ve yetişkin matrisler 12 ve yenilenmesini teşvik etme yeteneği moleküllerin durumu belirgin farklılıklar 13 araştırılmıştır. Hücresizleştirilmiş yetişkin matrisler hücre yeniden popüle bir ECM iskele ve biyosentetik bir kalp üretimi temin etmek üzere çeşitli çalışmalarda kullanılmıştır. kök hücre teknolojileri bu çalışmalar ve yeni keşifler, hızla ilerleyen ederken, birkaç engeller yerine getirilmesi için henüz. Örneğin, matris duvara matrisin doğal yapısını, hücre entegrasyon korunmasında sınırlamalar ve yeteneği bu yaklaşımın başarısı çoğalmasına ve büyüme tüm sınır desteklemek. Üstün rejeneratif özellikler yenidoğan kalp atfedilen olsa da, böyle bir doku kullanarak pratik yönleri onun keşif sınırlıdır. Yenidoğan kalp gösterdi rejeneratif kapasitesi dayanarak, bir geliştirerek yeni matris geliştirdikP3 fare kalp için Decellularization tekniği. Daha önce 6 belirlenir, ancak kalp hasat, decellularize ve recellularize için yeterince büyük olduğu gibi kardiyak rejenerasyon pencere içinde olduğu gibi P3 kalp bu çalışmalar için seçildi. Bu çalışmanın amacı, bir yenidoğan fare kalp bir matris oluşturmak fizibilite göstermektir. Çalışmalarımız ECM yapısal ve proteinli bütünlüğünü koruyarak bir dakika, yenidoğan kalp decellularizing fizibilitesi için kanıt. Biz de MCherry ifade eden kardiyomiyositlerinin bu kalp ECM recellularize yeteneğini göstermek ve recellularization aşağıdaki çeşitli kardiyak belirteçlerin ifadesi için bu kardiyomiyositlerde inceledik. Bu teknoloji, bir biyosentetik kalbin gelişiminde neonatal matrisin üstünlük denemeden geçirilmesine izin verecektir.

<table border="0" cellpadding="0" cellspacing="0" width="1054">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">1. Materials For Mouse Heart Isolation</td>
			<td style="width:323px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)</td>
			<td>Jackson Laboratories</td>
			<td>21577</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm Culture dish</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline pH 7.4 (sterile)</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Use Blade</td>
			<td>Stanley</td>
			<td>28-510</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Scissors</td>
			<td>Moria Bonn (Fine Science Tools)</td>
			<td>14381-43</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors 10 cm</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5 Forceps</td>
			<td>Dumnot (Fine Science Tools)</td>
			<td>11295-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Materials For Decellularization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inlet adaptor</td>
			<td>Chemglass</td>
			<td>CG-1013</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Septum</td>
			<td>Chemglass</td>
			<td>CG-3022-99</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/8 in. ID x 3/8 OD C-Flex tubing</td>
			<td>Cole-Parmer&#160;</td>
			<td>EW-06422-10</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K33</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K26</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolene 7-0 surgical suture&#160;</td>
			<td>Ethicon</td>
			<td>8648G</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring stand</td>
			<td>Fisher Scientific</td>
			<td>S47807</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp</td>
			<td>Fisher Scientific</td>
			<td>05-769-6Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp regular holder</td>
			<td>Fisher Scientific</td>
			<td>05-754Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 cc syringe barrel&#160;</td>
			<td>Coviden</td>
			<td>1186000777T</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beaker</td>
			<td>Kimble</td>
			<td>14000250</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22g x 1 Syringe Needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 cc syringe&#160;</td>
			<td>Coviden</td>
			<td>8881512878</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stop cock&#160;&#160;</td>
			<td>Smith Medical&#160;</td>
			<td>MX5311L</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22 x 1 g needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE50 tubing&#160;</td>
			<td>BD Clay Adams Intramedic</td>
			<td>427411</td>
			<td>Must be formable by heat. Polyethylene recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% SDS&#160;</td>
			<td>Invitrogen</td>
			<td>15525-017</td>
			<td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Make up fresh solution from a 10% stock and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile dH2O</td>
			<td>Hyclone</td>
			<td>SH30538.02</td>
			<td>Or MilliQ system purified water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Pen/Strep&#160;</td>
			<td>Corning CellGro</td>
			<td>30-001-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3. Materials For DNA Quantitation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K&#160;</td>
			<td>Fisher</td>
			<td>BP1700</td>
			<td>&#62;30U/mg activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl*6H2O</td>
			<td>Mallinckrodt</td>
			<td>5958-04</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base/hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T1503/T5941</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pico-Green dsDNA assay kit</td>
			<td>Life Technologies&#160;</td>
			<td>P7589</td>
			<td>requires fluorimeter to read</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4. Method for fixation and sectioning of tissue.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin Type A from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td>must be 300 bloom or greater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5. Method for tissue histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomolds 10 x 10 x 5mm</td>
			<td>Tissue-Tek</td>
			<td>4565</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Hacker/Bright</td>
			<td>Model OTF</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides&#160; 25 x 75 x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-550-19</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin 560&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801570</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Define</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3803590</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue buffer&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3802915</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcoholic Eosin Y 515&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801615</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formula 83 Xylene substitute&#160;</td>
			<td>CBG Biotech&#160;</td>
			<td>CH0104B</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount Mounting Medium&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>SP15-500</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Antibody</td>
			<td>Rockland</td>
			<td>600-401-106.1</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Actinin Antibody</td>
			<td>Abcam</td>
			<td>AB9465</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mCherry Antibody</td>
			<td>Abcam</td>
			<td>AB205402</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NKX2.5 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-8697</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-mouse AF488 Antibody</td>
			<td>Life Technology</td>
			<td>&#160;A21202&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-chicken AF594 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>703-585-155&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-goat CY5 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>705-175-147</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>111-587-003&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (Ca, Mg Free)</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Corning CellGro</td>
			<td>25-060-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primeria 100 mm plates</td>
			<td>Corning</td>
			<td>353803</td>
			<td>Primeria surface enhances fibroblast attachment promoting a higher myocyte purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Difco</td>
			<td>215240</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase II</td>
			<td>Sigma-Aldrich</td>
			<td>D8764</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Delbecco&#39;s Minimal Essential Media)</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vitamin B12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V6629</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin coated plates&#160;</td>
			<td>BD Bioscience</td>
			<td>354501</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heart bioreactor glassware</td>
			<td>Radnoti Glass Technology</td>
			<td>120101BEZ</td>
			<td>Must be sterilizable by autoclaving or gas.</td>
		</tr>
	</tbody>
</table>

neonatal cardiac scaffolds: novel matrices for regenerative studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

decellularization Ortalama olarak, bu protokolü kullanarak bir P3 kalp Decellularization zamanı yaklaşık 14 saattir. P3 yenidoğan için 23 mg, ortalama kalp ağırlığı verilmiş. Acellularity Şekil 3a tamamen bozulmamış P3 yenidoğan kalp (bütün montaj). Şekil 3b. Decellularization aşağıdaki aynı kalp gösterir 4a Şekiller ve s...

Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Bu çalışmalarda, biz rejeneratif çalışmalarda kullanılmak üzere yeni, yenidoğan murin kardiyak iskeleleri için bir yöntem sağlamak.

Yenidoğan Kardiyak iskeleleri: Rejeneratif Çalışmaları Roman Matrisler

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

neonatal-cardiac-scaffolds-novel-matrices-for-regenerative-studies

Research

JoVE Journal

Bioengineering

7.7K Görüntüleme.  University of Minnesota.  Bu çalışmalarda, biz rejeneratif çalışmalarda kullanılmak üzere yeni, yenidoğan murin kardiyak iskeleleri için bir yöntem sağlamak. 

Video: Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Hücresel Daralma miktarının Şikayet Matrisler hazırlanması

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

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 ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Yoğunluk Gradient Katmanlı Polimerizasyonu (DGMP): Multi-bölmesi oluşturma için A Novel Tekniği, Doku Mühendisliği için Özelleştirilebilir İskelesi

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Fizyolojik Akış Koşullarında Sirkülasyon Hücre Kemokin-yönettiği Ekstravazasyon Eğitim Bir Roman Üç boyutlu Akış Odası Cihazı

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Rejeneratif Araştırma harmonik nanopartiküller

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Fiber Teşkilatı geçişleri ile electrospun Nanolif iskeleleri

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Tanımlı İnsan Engineered Kalp Dokuların inşaatı Kardiyak Hücre Tedavisinin Mekanizmaları Eğitim için

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Şeffaf olmayan iskelelerinin üzerinde Görüntüleme Hücre Canlılık - Roman Örme Titanyum İmplant Örnek Kullanımı

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Anatomik olarak gerçekçi yenidoğan kalp modeli kullanılmak üzere yenidoğan hasta simülatörleri

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

Matrisler roman işlemi 3D baskı için Decellularized

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...