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

Tutti gli esperimenti di topo sono stati eseguiti in conformità con Animal Welfare Act degli Stati Uniti e sono stati approvati dal Comitato di Cura e uso istituzionale degli animali presso l&#39;Università del Minnesota. 1. Metodo per l&#39;isolamento Cuore mouse <ol><li> Euthanize un mouse neonatale per decapitazione con una sola lama utilizzo. </li><li> Tampone torace con il 70% di etanolo. </li><li> Sezionare la pelle dal petto tagliando via dalla parete toracica con le forbici standard, tirando la pelle lateralmente una coppia di pinze # 5. </li><li> Perforare l&#39;addome appena inferiore allo sterno con le forbici tagliando attraverso la parete addominale. Afferrando il processo xifoideo con # 5 pinze, ritrarre lo sterno rostralmente dal corpo durante il taglio se le costole su entrambi i lati del torace con le forbici. La gabbia toracica si riflette superiormente con la pinza per rivelare il cuore. </li><li> Senza mezzi termini sezionare i due lobi principali del timo tirando lateralmente # 5 pinze, esponendol&#39;arco dell&#39;aorta, nonché le vene cavali e polmonari. </li><li> Transetto le principali arterie dall&#39;arco aortico e l&#39;aorta in sé, con le forbici a molla 10 cm. Conservare l&#39;aorta tra la base del cuore e l&#39;arteria anonima. Afferrare le estremità dei vasi mozzate con il # 5 pinze per riflettere cuore avanti, separandolo dalla trachea e dell&#39;esofago. </li><li> Transect vascolare polmonare e altre grandi vene, tagliando tra i polmoni e il cuore su entrambi i lati del cuore con le forbici a molla 10 cm. Le vene rimangono aperti per fornire il drenaggio. </li><li> Rimuovere il cuore dal mediastino con il # 5 pinze, afferrare le estremità mozzate dei grandi vasi. Mettere il cuore in un piatto di cultura 60 millimetri contenente sterile tampone fosfato salino (PBS) per cateterizzazione. </li></ol> 2. Metodo per Decellularization da Langendorff perfusione <ol><li> Preparare il complesso di catetere in anticipo. Disegnare una sezione di quattro centimetriPE 50 tubi in una piccola fiamma alcool per creare un più piccolo, più sottile catetere. Tagliare le estremità con una lama per soddisfare le dimensioni (circa. 300 um OD diametro esterno di una flangia di circa. OD 500 micron) di figura 1. Ciò fornirà due cateteri simmetriche da ciascun lato del tiro. </li><li> Puntare l&#39;estremità tagliata del tubo nella fiamma brevemente, per fondere una flangia sulla apertura all&#39;estremità sottile. </li><li> Riempire la siringa 12 ml con PBS e assemblare il catetere inserendo un rubinetto a 3 vie sulla siringa. Aggiungere un 22 G x 1 ago per il rubinetto e guidare l&#39;ago attraverso il setto. Far scorrere il tubo del catetere PE disegnato sul dell&#39;ago (Fig. 1). </li><li> Lavare le parti assemblate con PBS, assicurando che tutte le bolle d&#39;aria sono stati rimossi. </li><li> Inserire il catetere, preparato come descritto sopra, in aorta del cuore isolato, che non si estende oltre la valvola aortica e legare con un tirante di 7-0 sutura. Appoggiare la flangia di tegli catetere prossimale contro la cravatta, facendo una perfetta tenuta e prevenire il cuore di venire fuori il catetere. </li><li> Osservare la cateterizzazione sotto ingrandimento, mentre delicatamente perfusione cuore utilizzando la siringa contenente PBS. Assicurarsi che non vi siano perdite nel sistema e il tessuto blanches omogeneo come il sangue latente viene rimosso. </li><li> Posizionare il setto nel collo dell&#39;adattatore ingresso e sigillarlo ripiegando i lati sopra il vetro. </li><li> Fissare il serbatoio riempito con 60 ml di 1% solfato di sodio dodecil (SDS) in acqua distillata (DH 2 O) tramite una linea di lunghezza sufficiente a produrre una colonna che genera 20 millimetri Hg di pressione, come mostrato in Fig. 1. Calcolare la pressione dall&#39;alto colonna di liquido in base al rapporto di 1 mm Hg è pari a 1,3595 centimetri H 2 O. Attenzione: SDS è una polvere altamente flocculante con proprietà irritanti. Contatto dovrebbe essere evitato. Maneggiare la polvere utilizzando appropriati indumenti protettivi. </li><li> Profumato cuore con 1% SDS per 14 ore. Il cuore sarà traslucido in apparenza senza tessuto rimanente osservabile. </li><li> Lavare il sistema fino al rubinetto della soluzione rimanente SDS e sostituirla con 10 ml dH 2 O. Profumato cuore con 10 ml di 1% Triton X-100 (diluito in acqua distillata), seguita da 10 ml dH 2 O, seguiti da 60 ml di PBS contenenti 1x streptomicina penicillina (Pen-Strep). </li><li> Conservare il cuore in PBS con 1x Pen-Strep a 4 ° C. Se l&#39;applicazione palo decellularization destinato richiede perfusione quindi il catetere in aorta deve essere mantenuto, altrimenti tagliare il legame di sutura e rimuovere il catetere. </li></ol> Determinazione 3. DNA <ol><li> Preparare un digest del decellularized ECM o il controllo cardiaco utilizzando 200 mg / ml proteinasi K in 50 mM KCl, 2,5 mM MgCl 2, 0,45% di Tween 20 in 10 mM Tris (pH 8,3). </li><li> Incubare il tessuto a 55 ° C con agitazione fino a quando il tessuto è dissolto,in genere 4-5 ore. </li><li> Quantificare il contenuto di DNA della omogenato con un DNA test di 4,11 vincolante. </li></ol> 4. Fissazione e il sezionamento di tessuto <ol><li> Fix decellularized, cuori recellularized o controllo P3 in 4% paraformaldeide in PBS per 30 minuti a temperatura ambiente. </li><li> Lavare il tessuto in PBS 3x. </li><li> Posizionare il tessuto in una soluzione di 7,5% di saccarosio in tampone fosfato 0,1 M a 4 ° C fino affonda. </li><li> Cambiare la soluzione al 15% di saccarosio in tampone fosfato 0,1 M a 4 ° C, ancora, fino affonda. </li><li> Riscaldare il tessuto a 37 ° C, sostituire la metà del volume con soluzione di gelatina nel 15% di saccarosio e 0,1 M tampone fosfato ed equilibrare la notte. Le concentrazioni di gelatina sono aumentati graduale attraverso 1%, 2,5%, 5% e infine 7,5% due volte. </li><li> Mettere i campioni in cryomolds utilizzando fresco 7,5% di gelatina. Per ripristinare la forma dei campioni decellularized, liquido gelatina può essere perfuso nelle camere come tlui cuori sono modellati. Congelare galleggiando i cryomolds in azoto liquido. Conservare a -80 ° C. </li><li> CUT (10 micron di spessore) sezioni con un criostato. Portare la slitta vicino alla superficie della sezione e osservare la sezione spostarsi fuori dal coltello sulla superficie dovuta alla carica sui vetrini trattati elettrostaticamente. Asciugare le diapositive durante la notte e conservare a -80 ° C per le analisi future. </li><li> Rimuovere i vetrini dal congelatore, equilibrare a temperatura ambiente e poi posto in una vaschetta Coplin contenente PBS per 20 minuti a 37 ° C per sciogliere la gelatina. </li><li> Macchiare le sezioni tagliate dal punto 4.7 con ematossilina eosina. Collocare i vetrini in una vaschetta Coplin. Esporre le diapositive idratato al punto 4.8 per ematossilina per 45 secondi, l&#39;acqua del rubinetto per 1,5 minuti, tampone per 3 minuti, l&#39;acqua del rubinetto per 1 minuto, agente azzurrante per 1,5 min, 80% di etanolo per 1 min, alcolica Eosina Y per 8 secondi, 80% di etanolo per 1 minuto, 100% di etanolo per 1 min 2x, stanza di compensazione (sostituto di xilene) per 1 min 3x, coprioggetto con resinaMezzo di montaggio basato. Esaminare le diapositive al microscopio. </li><li> Per confermare il mantenimento di ECM proteine ​​reattività del ECM cuore, macchia per proteine ​​strutturali ECM come il collagene IV, ponendo le diapositive idratati in una camera umidificata e trattare con il 10% normale asino siero (NDS) in tampone fosfato con 0,1% Triton -X100 (PBST) per 1 ora a temperatura ambiente (RT). Utilizzare un volume sufficiente a coprire le sezioni di tessuto. </li><li> Sostituire la soluzione con l&#39;anticorpo primario di scelta ad una diluizione empiricamente determinato 5% NDS / PBST. Per esempio, l&#39;anticorpo collagene IV è stato diluito a 1: 150. Incubare per una notte a 4 ° C. </li><li> Lavare i vetrini con PBST 3x e applicare un anticorpo secondario di scelta coniugato con un colorante fluorescente. Diluire l&#39;anticorpo di una quantità determinata empiricamente. Incubare per 1 ora a RT. Lavare con PBS 3x. </li><li> Colorare i vetrini con un colorante legame al DNA come DAPI per verificare l&#39;assenza di nuclei delle cellule utilizzando un mounti DAPI contenenteng media quando il coperchio scivolare le diapositive. </li><li> Esaminare i vetrini con un microscopio a fluorescenza da 50 a 400X. </li></ol> 5. P1 neonatale murino ventricolare cardiomiociti per Recellularization <ol><li> Spruzzare ogni cucciolo con il 70% di etanolo e decapitarlo con una lama singola uso. </li><li> Tenere ogni pup tra il pollice e l&#39;indice, mentre il torace è diviso sulla linea mediana con piccole forbici sterili per esporre la cavità toracica. Applicare pressione per consentire cuore di sporgere liberamente dal petto mentre è tagliato libera dei grandi vasi e gli atri lasciando solo il tessuto ventricolare. </li><li> Mettere ogni cuore direttamente in un tubo da 50 ml contenente 20 ml ghiacciata di calcio, soluzione salina tamponata Bicarbonato libero di Hank con 20 mM 4- (2-idrossietil) Acido -1-piperazineethanesulfonic (CBFHH) fino a quando tutti i cuori sono raccolti. </li><li> Aspirare il CBFHH e aggiungere 10-15 ml di CBFHH fresco (ghiacciata) al tubo. Lavare il tessuto agitando il tessuto in tegli provetta diverse volte. </li><li> Decantare la soluzione contenente il cuore in una plastica Petri piastre da 60 mm. </li><li> Sezionare qualsiasi tessuto atriale latente dal cuore sotto ingrandimento sul ghiaccio da tagliare via dal cuore con le forbici a molla. Tritare i ventricoli in piccoli pezzi in modo omogeneo dimensioni. Rimuovere qualsiasi tessuto non-ventricolare separato dal piatto con # 5 pinze. </li><li> Pipettare tanto della soluzione CBFHH come è necessario per trasferire i frammenti di tessuto in una piccola fiala sterile contenente una sterile bar micro mescolare. </li><li> Lasciare il tessuto sedimentare sul fondo della fiala e rimuovere la soluzione CBFHH. </li><li> Portare 50 ml di una soluzione enzimatica 1,75 mg / ml di tripsina, 20 ug / ml DNasi II in CBFHH. Dispensare 5 ml di questa soluzione enzimatica e aggiungerlo a una provetta contenente il tessuto ventricolare tritato e posto su un agitatore magnetico. Mescolare ad un tasso costante (340 rpm) a temperatura ambiente per 8 min. NOTA: L&#39;azione di agitazione deve essere dolce e tuttavia consentireper la sospensione della sospensione. </li><li> Rimuovere la fiala dalla piastra mescolare e lasciare l&#39;impasto per sedimentare per 3 min. Pipetta e scartare il surnatante iniziale di digerire, in quanto contiene principalmente le cellule del sangue e detriti dei tessuti. </li><li> Pipettare un secondo 5 ml aliquota della soluzione enzimatica e aggiungerlo al ventricolo digerire. Mescolare per 8 minuti e lasciare sedimentare per altri 3 minuti. Prima di iniziare i digest, preparare due provette da 50 ml (etichettati 1 e 2) ciascuna contenente 12 ml di ghiacciata siero fetale bovino (FBS). Mettere un filtro cella di 40 micron in cima a ciascun tubo. Pipettare questo surnatante attraverso il filtro sul tubo 1. </li><li> Ripetere il processo di digestione ulteriori 8 volte, alternando il posizionamento del surnatante digest tra i due tubi contenenti FBS. Dividere l&#39;ultimo surnatante in parti uguali tra i tubi 1 e 2 per garantire volumi uguali in ogni tubo. NOTA: Ogni tubo di raccolta conterrà ora 32,5 ml di volume totale di sospensione cellulare. </li><li> Centrifugare le provette contengonoing XG surnatante 150 per 6 minuti a 4 ° C. </li><li> Mentre la raccolta delle aliquote di digestione, preparare cinque 100 piastre di coltura dei tessuti mm con 6 ml di media (Dulbecco Modified Eagle media (DMEM) con il 10% FBS, 1x Pen-Strep, 1x L-glutammina) per piastra. Incubare le aliquote a 37 ° C, 5% CO 2 per 30 minuti per equilibrare la media. </li><li> Aspirare la miscela di enzimi CBFHH e risospendere le cellule in 30 ml di terreni di coltura preparato in precedenza, che unisce le cellule da entrambi i tubi di raccolta. </li><li> Ritorno 6 ml di sospensione cellulare per ciascuna delle piastre 100 mm e incubare per 45 min a 37 ° C. </li><li> Al termine dell&#39;incubazione, trasferire le cellule non aderenti a 5 piatti nuovi e ri-incubare per 45 min (la frazione fibroblasti avrà iniziato ad aderire ai piatti e può essere coltivata separatamente se desiderato). </li><li> Al termine del secondo turno di pre-placcatura, raccogliere i media e le cellule non aderenti dai piatti e far girare i mezzi di comunicazione a 150xg per 6 min. </li><li> Aspirare supporti in eccesso per portare le cellule alla concentrazione desiderata approssimativa, (4.0 x 10 5 cellule per costrutto in 100 ml di perfusato). Rimuovere una aliquota per il conteggio e la vitalità o il test di 14. </li></ol> 6. bioreattore Recellularization di P3 Cuore Matrix <ol><li> Pretrattare la matrice cuore isolato con terreni di coltura (vedi punto 5.15) per una notte prima di aggiungere le cellule. </li><li> Assemblare gli elementi di un sistema di Langendorff come mostrato in Fig. 2. Autoclave le parti in vetro e ossido di etilene sterilizzare le parti in plastica per assicurare la sterilità. Varie unità secondarie del sistema possono essere assemblati in una cappa a flusso laminare prima di essere montato sul supporto. Di circolazione bagno di acqua e riscaldata percorso del flusso d&#39;acqua è stato omesso per chiarezza. </li><li> Riempire il sistema assemblato con 120 ml di terreni di coltura tissutale (vedere il punto 5.15). </li><li> Posizionare la matrice cuore cateterismo dal punto 2.14 in un cul 60 millimetripiatto tura sotto ingrandimento e collegare una siringa da 1 ml con 22 G x 1 ago caricato con sospensione cellulare dal punto 5.20 al catetere. Assicurarsi che questa assemblea è privo di bolle d&#39;aria per evitare embolizzazione cuore. </li><li> profumato delicatamente i 100 ml di sospensione cellulare nella matrice cuore attraverso le arterie coronarie. Una volta completato, staccare la combinazione della siringa / ago. NOTA: Un lento perfusione di circa mq. 20 ml per min è necessaria per evitare che le cellule di fluire fuori dalle vene, in transito cuore. </li><li> Con un 22g stub smussato fissato al raccordo Luer sul lato inferiore del coperchio vaso cuore bioreattore, spingere il catetere sul perno. </li><li> Avviare la pompa peristaltica e osservare che il flusso procede come illustrato in figura 2. Flusso procede dal serbatoio tramite la pompa attraverso il gorgogliatore al catetere posizionato in aorta nel passo 2.5 (percorso rosso in Fig. 2). Osservare il flusso della circolazione cardiaco della venas e gocciolare dall&#39;apice, ricircolo al serbatoio media. NOTA: La portata perfusione dipende da fattori individuali quali la resistenza vascolare del costrutto, e la dimensione del cuore, ma un tasso di 50 a 100 microlitri / min è un buon punto di partenza. In momenti intermedi, le valutazioni funzionali possono essere eseguiti. La scala dimensione del cuore recellularized neonatale limita le valutazioni, che possono essere eseguite, ma abbiamo stabilito che i sistemi basati otticamente possono essere utilizzati per quantificare battere comportamento così come sono stati utilizzati in cuori di ratto adulto. 4 Il costrutto può essere perfusi per esteso periodi di tempo (fino a 23 ore). </li></ol>

<ol>
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The authors have nothing to disclose.

La dipendenza di questa tecnica su ripetuto perfusione del cuore fa evitare un&#39;embolia una componente critica di un esito positivo. Dal cateterizzazione iniziale del cuore nei passaggi 2.2-2.6, ai cambiamenti di soluzione tra Piazza 2.8-2.14, ci sono manipolazioni che possono permettere l&#39;introduzione di bolle d&#39;aria che compromettono il flusso di perfusato nel miocardio. A causa del peso ridotto cuore neonatale, anche bolle minute vascolarizzazione possono causare un infarto tecnica, rendendo il decellulari...

L&#39;insufficienza cardiaca è comune e mortale. È una malattia progressiva che si traduce in una riduzione della contrattilità del cuore, che ostacola il flusso di sangue agli organi e lascia le richieste metaboliche del corpo insoddisfatte. Si stima che 5,7 milioni di americani hanno l&#39;insufficienza cardiaca ed è la causa primaria di ospedalizzazione negli Stati Uniti 9. Il costo collettivo di trattamento di pazienti in insufficienza cardiaca negli Stati Uniti supera i $ 300 miliardi di dollari l&#39;anno 9-10. L&#39;unica terapia definitiva per l&#39;insufficienza cardiaca allo stadio terminale è il trapianto di cuore ortotopico. Ogni anno, si stima che più di 100.000 cuori dei donatori sono necessari per le procedure di trapianto cardiaco negli Stati Uniti 1-2. A causa del limitato numero di donatori, solo circa 2.400 trapianti vengono effettuati ogni anno negli Stati Uniti 2. Chiaramente, questa carenza di organi deve essere affrontato come altre strategie sono tenuti a produrre organi aggiuntivi per il trapiantozione e, idealmente, questi organi sarebbe autologo in modo da evitare le complicazioni associate con il rifiuto e la durata immunosoppressione. Cardiomiociti adulti mammiferi dimostrano una limitata capacità di rigenerazione su di infortunio, ma recenti evidenze suggeriscono che mammiferi cuori neonatali mantengono una notevole capacità rigenerativa dopo un trauma 5-8. In particolare, dopo la resezione chirurgica parziale, una finestra rigenerativa è stato scoperto tra la nascita e postnatale giorno 7. Questo periodo rigenerativa è caratterizzata da una mancanza di cicatrice fibrotica, formazione di neovascolarizzazione, rilascio di fattori angiogenici dalla epicardio e cardiomiociti proliferazione 5-8 , 11. Questa finestra rigenerativo di tempo fornisce il potenziale di utilizzo cuore neonatale quale nuovo fonte di materiale per lo sviluppo di un cuore bioartificiale. La matrice extracellulare è nota per fornire spunti importanti per promuovere cardiomyocyte la proliferazione e la crescita. Chiare differenze nella disponibilità di molecole nelle matrici neonatali e adulti 12 e la loro capacità di promuovere la rigenerazione sono state esplorate 13. matrici adulti decellularized sono stati utilizzati in diversi studi per fornire un ponteggio ECM per ripopolazione cellulare e la generazione di un cuore bioartificiale. Mentre questi studi, e le nuove scoperte nel campo delle tecnologie di cellule staminali, stanno avanzando rapidamente, diversi ostacoli devono ancora essere soddisfatte. Ad esempio, limitazioni nella preservando la struttura originaria della matrice, l&#39;integrazione cellulare nella parete della matrice, e la capacità di sostenere la proliferazione e la crescita tutti i limiti del successo di questo approccio. Mentre gli attributi rigenerativa superiori sono state attribuite al cuore neonatale, gli aspetti pratici di utilizzo di un tessuto così hanno limitato la sua esplorazione. Sulla base della capacità rigenerativa dimostrata del cuore neonatale, abbiamo sviluppato nuovi matrici sviluppando unatecnica di decellularization per il cuore P3 mouse. Il cuore P3 è stato scelto per questi studi in quanto è all&#39;interno della finestra di rigenerazione cardiaca come precedentemente determinato 6 ma il cuore è grande abbastanza per il raccolto, decellularize e recellularize. L&#39;obiettivo di questo studio è quello di dimostrare la fattibilità della creazione di una matrice da un cuore neonatale del mouse. I nostri studi forniscono prove per la fattibilità di decellularizing un minuto, cuore neonatale pur mantenendo l&#39;integrità strutturale e proteica della ECM. Dimostriamo anche la capacità di recellularize questo ECM cardiaco con mCherry cardiomiociti che esprimono e abbiamo esaminato questi cardiomiociti per l&#39;espressione di diversi marcatori cardiaci seguenti Recellularization. Questa tecnologia permetterà per la prova della superiorità di una matrice neonatale per lo sviluppo di un cuore bioartificiale.

<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 In media, il tempo di decellularization di un cuore P3 utilizzando questo protocollo è di circa 14 ore. dato un peso medio di 23 mg di cuore per il neonato P3. Acellularity Figura 3a mostra un cuore neonatale completamente intatto P3 (tutto il monte). La figura 3b mostra lo stesso cuore seguente decellularization. Figure 4a e 4b</strong...

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

In questi studi, forniamo metodologia per nuovi, neonatali, impalcature cardiaci murini per l&#39;uso in studi rigenerative.

Neonatale cardiaco Impalcature: Matrici innovativi per gli Studi rigenerativi

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

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Research

JoVE Journal

Bioengineering

7.7K Views.  University of Minnesota.  In questi studi, forniamo metodologia per nuovi, neonatali, impalcature cardiaci murini per l'uso in studi rigenerative. 

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.

Preparazione di matrici di reclamo per Quantificare Contrazione Cellular

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

Ricerca

Bioingegneria

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.

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

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

Gradiente di densità multistrato polimerizzazione (DGMP): una tecnica innovativa per la creazione di comparti multipli, Ponteggi personalizzabili per l&#39;ingegneria tissutale

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.

A Novel tridimensionale del flusso Camera dispositivo per studiare Chemokine-diretto stravaso di cellule circolanti in condizioni di flusso fisiologici

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.

Harmonic Nanoparticelle for Regenerative ricerca

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.

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

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.

Costruzione di Engineered Definito umani cardiache tessuti per studiare i meccanismi della terapia cellulare cardiaca

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.

Viabilità imaging cellulare su scaffold non trasparenti - con l&#39;esempio di un romanzo a maglia Titanium Implant

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.

Modello di cuore neonatale anatomicamente realistici per l'uso in simulatori paziente neonatale

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.

Processo innovativo per la stampa 3D Decellularized matrici

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