Name: neonatal cardiac scaffolds: novel matrices for regenerative studies
Uploaded: 2016-11-05T14:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

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>

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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><tbody><tr><td colspan="4">&lt;strong&gt;1. Materials for mouse heart isolation&lt;/strong&gt;</td></tr><tr><td>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><td>60 mm Culture dish</td><td>BD Falcon</td><td>353004</td><td>or equivalent</td></tr><tr><td>Phosphate buffered saline pH 7.4 (sterile)</td><td>Hyclone</td><td>SH30256.01</td><td>or equivalent</td></tr><tr><td>Single Use Blade</td><td>Stanley</td><td>28-510</td><td>or equivalent</td></tr><tr><td>Standard Scissors</td><td>Moria Bonn (Fine Science Tools)</td><td>14381-43</td><td>or equivalent</td></tr><tr><td>Spring Scissors 10 cm</td><td>Fine Science Tools</td><td>15024-10</td><td>or equivalent</td></tr><tr><td>Vannas Spring Scissors - 3 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-00</td><td>or equivalent</td></tr><tr><td>#5 Forceps</td><td>Dumnot (Fine Science Tools)</td><td>11295-00</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;2. Materials for decellularization&lt;/strong&gt;</td></tr><tr><td>Inlet adaptor</td><td>Chemglass</td><td>CG-1013</td><td>autoclavable</td></tr><tr><td>Septum</td><td>Chemglass</td><td>CG-3022-99</td><td>autoclavable</td></tr><tr><td>1/8 in. ID x 3/8 OD C-Flex tubing</td><td>Cole-Parmer</td><td>EW-06422-10</td><td>autoclavable</td></tr><tr><td>Male luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K33</td><td>autoclavable</td></tr><tr><td>Female luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K26</td><td>autoclavable</td></tr><tr><td>Prolene 7-0 surgical suture&amp;nbsp;</td><td>Ethicon</td><td>8648G</td><td>or equivalent</td></tr><tr><td>Ring stand</td><td>Fisher Scientific</td><td>S47807</td><td>or equivalent</td></tr><tr><td>Clamp</td><td>Fisher Scientific</td><td>05-769-6Q</td><td>or equivalent</td></tr><tr><td>Clamp regular holder</td><td>Fisher Scientific</td><td>05-754Q</td><td>or equivalent</td></tr><tr><td>60 cc syringe barrel&amp;nbsp;</td><td>Coviden</td><td>1186000777T</td><td>or equivalent</td></tr><tr><td>Beaker</td><td>Kimble</td><td>14000250</td><td>or equivalent</td></tr><tr><td>22 G x 1 Syringe Needle</td><td>BD</td><td>305155</td><td>or equivalent</td></tr><tr><td>12 cc syringe</td><td>Coviden</td><td>8881512878</td><td>or equivalent</td></tr><tr><td>3-way stop cock</td><td>Smith Medical</td><td>MX5311L</td><td>or equivalent</td></tr><tr><td>PE50 tubing</td><td>BD Clay Adams Intramedic</td><td>427411</td><td>Must be formable by heat. Polyethylene recommended.</td></tr><tr><td>1% SDS</td><td>Invitrogen</td><td>15525-017</td><td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.</td></tr><tr><td>1% Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td>Make up fresh solution from a 10% stock and filter sterilize before use.&amp;nbsp;</td></tr><tr><td>Sterile dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Hyclone</td><td>SH30538.02</td><td>Or MilliQ system purified water.</td></tr><tr><td>1x Pen/Strep</td><td>Corning CellGro</td><td>30-001-Cl</td><td>or equivalent&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;3. Materials for DNA quantitation&lt;/strong&gt;</td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700</td><td>&gt;30 U/mg activity</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>or equivalent</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mallinckrodt</td><td>5958-04</td><td>or equivalent</td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1379</td><td>or equivalent</td></tr><tr><td>Tris base/hydrochloride</td><td>Sigma-Aldrich</td><td>T1503/T5941</td><td>or equivalent</td></tr><tr><td>Pico-Green dsDNA assay kit</td><td>Life Technologies&amp;nbsp;</td><td>P7589</td><td>requires fluorimeter to read</td></tr><tr><td colspan="4">&lt;strong&gt;4. Method for fixation and sectioning of tissue&lt;/strong&gt;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>or equivalent</td></tr><tr><td>Gelatin Type A from porcine skin</td><td>Sigma-Aldrich</td><td>G2500</td><td>must be 300 bloom or greater</td></tr><tr><td colspan="4">&lt;strong&gt;5. Method for tissue histology&lt;/strong&gt;</td></tr><tr><td>Cryomolds 10 x 10 x 5 mm</td><td>Tissue-Tek</td><td>4565</td><td>or equivalent</td></tr><tr><td>Cryostat</td><td>Hacker/Bright</td><td>Model OTF</td><td>or equivalent</td></tr><tr><td>Microscope Slides&amp;nbsp; 25 x 75 x 1 mm</td><td>Fisher Scientific</td><td>12-550-19</td><td>or equivalent</td></tr><tr><td>Hematoxylin 560&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801570</td><td>or equivalent</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2701</td><td>or equivalent</td></tr><tr><td>Define</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3803590</td><td>or equivalent</td></tr><tr><td>Blue buffer&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3802915</td><td>or equivalent</td></tr><tr><td>Alcoholic Eosin Y 515&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801615</td><td>or equivalent</td></tr><tr><td>Formula 83 Xylene substitute&amp;nbsp;</td><td>CBG Biotech&amp;nbsp;</td><td>CH0104B</td><td>or equivalent</td></tr><tr><td>Permount Mounting Medium&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>SP15-500</td><td>or equivalent</td></tr><tr><td>Collagen IV Antibody</td><td>Rockland</td><td>600-401-106.1</td><td>or equivalent</td></tr><tr><td>&amp;alpha;-Actinin Antibody</td><td>Abcam</td><td>AB9465</td><td>or equivalent</td></tr><tr><td>mCherry Antibody</td><td>Abcam</td><td>AB205402</td><td>or equivalent</td></tr><tr><td>NKX2.5 Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-8697</td><td>or equivalent</td></tr><tr><td>Donkey anti-mouse AF488 Antibody</td><td>Life Technology</td><td>A21202</td><td>or equivalent</td></tr><tr><td>Donkey anti-chicken AF594 Antibody</td><td>Jackson Immunoresearch</td><td>703-585-155&amp;nbsp;</td><td>or equivalent</td></tr><tr><td>Donkey anti-goat CY5 Antibody</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>705-175-147</td><td>or equivalent</td></tr><tr><td>Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td><td>Jackson Immunoresearch</td><td>111-587-003</td><td>or equivalent</td></tr><tr><td>Prolong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36930</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;6. Isolation of neonatal ventricular cardiomyocytes using pre-plating&lt;/strong&gt;</td></tr><tr><td>HBSS (Ca, Mg Free)</td><td>Hyclone</td><td>SH30031.02</td><td>or equivalent</td></tr><tr><td>HEPES (1 M)</td><td>Corning CellGro</td><td>25-060-Cl</td><td>or equivalent</td></tr><tr><td>Cell Strainer</td><td>BD Falcon</td><td>352340</td><td>or equivalent</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>or equivalent</td></tr><tr><td>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><td>Trypsin</td><td>Difco</td><td>215240</td><td>or equivalent</td></tr><tr><td>DNase II</td><td>Sigma-Aldrich</td><td>D8764</td><td>or equivalent</td></tr><tr><td>DMEM (Delbecco&#039;s Minimal Essential Media)</td><td>Hyclone</td><td>SH30022.01</td><td>or equivalent</td></tr><tr><td>Vitamin B12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>V6629</td><td>or equivalent</td></tr><tr><td>Fibronectin coated plates&amp;nbsp;</td><td>BD Bioscience</td><td>354501</td><td>or equivalent</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Hyclone</td><td>SH30910.03</td><td>or equivalent</td></tr><tr><td>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 ...

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

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

Capillary Force Lithography for Cardiac Tissue Engineering

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

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

Capillare Forza litografia per Cardiac Tissue Engineering

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

Ricerca

Bioingegneria

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingegneria dei tessuti: Costruzione di un multicellulare 3D Ponteggio per la consegna di Layered fogli Cellulari

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabbricazione di miogenici Costrutti di ingegneria tessutale

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Biologia

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Paragonabile decellularizzazione degli espianti di tessuto cardiaco fetali ed adulti come 3D-come le piattaforme per studi In Vitro

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

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

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

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

Un metodo basato su stampo netto della creazione senza impalcatura tridimensionale del tessuto cardiaco

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

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabbricazione di scaffold decellularizzati matrice della cartilagine-derivato

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparazione di tessuti cardiaci ingegnerizzati a forma di mesh derivati da cellule iPS umane per la riparazione miocardia in Vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Generazione, screening ad alto rendimento e biobanca di sferoidi cardiaci derivati da cellule staminali pluripotenti indotte dall'uomo

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

Generazione 3D di tessuto miocardico umano mediante scrittura di elettrofilatura per fusione di scaffold di policaprolattone e cellule cardiache derivate da hiPSC

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Trapianto di cuore in vivo per studiare la transizione da endotelio a mesenchimale

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Autore in primo piano: Un modello di trapianto di cuore di ratto eterotopico neonatale per lo studio della transizione da endotelio a mesenchimale  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Un modello di trapianto di cuore di ratto eterotopico neonatale per lo studio della transizione da endoteliale a mesenchimale

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...