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

Questo protocollo segue le linee guida LUMC dell&#39;animale comitato etico della ricerca. 1. Preparazione degli strumenti, Cultura Media e MTC Nota: eseguire tutti i preparativi in ​​cappa a flusso laminare. La MTC camera di perfusione, trappola per bolle e supporto sono descritti in Lieber et al., 2010 11. <ol><li> Disinfettare le pinze e forbici micro con il 70% di etanolo. Preparare una siringa da 5 ml con 21G ago con tampone (130 NaCl sterile Tyrode mM, 5,4 mM KCl, 6.0 mM Hepes, 1.2 MgSO mm 4, 1,2 mm KH 2 PO 4, 20 Glucosio mM, 1,5 mM CaCl 2 .2H 2 O, pH = 7.2). Preparare una siringa da 5 ml con ago 21G con la soluzione KCl sterile (100 mm). </li><li> Preparare 65 ml di terreno per cuore: DMEM supplementato con 10% siero fetale bovino, antibiotici / antimicotici (A / A; 100 unità di penicillina, 100 mg di streptomicina, e 0,25 ug di amfotericina B / ml), insulino-Transferrin-Selenio (ITS; 10 mg / ml di insulina, 5.5 mg / ml transferrina, 6.7 ng / ml selenito di sodio) e filtro sterilizzare. </li><li> Sterilizzare la camera di perfusione e gorgogliatore spruzzando con il 70% di etanolo e asciugare con carta assorbente. Assemblare la MTC (Figura 1D), tuttavia, inizialmente senza la camera di perfusione. Posizionare la trappola bolla sul cavalletto. Utilizzare una pompa peristaltica a rulli con testate pompa facile carico. <ol><li> Utilizzando tubo in silicone effettuare le connessioni tra il serbatoio (tubo da 50 ml) e la pompa, la pompa e il gorgogliatore, il gorgogliatore e serbatoio (Figura 1D). Rendere il tubo all&#39;interno dell&#39;incubatrice abbastanza lungo da permettere medio raggiungimento dell&#39;equilibrio gas con il gas nell&#39;incubatrice attraverso il tubo di gas permeabile silicio (1 m) e il tubo all&#39;esterno dell&#39;incubatrice più breve possibile. </li></ol></li><li> Riempire il tubo da 50 ml con il 70% di etanolo, accendere la pompa (velocità del flusso intorno a 1 ml / min per consentire la corretta circolazione) e lasciare che il CIR etanolocolare per 30 min per sterilizzare tutti i tubi. Rimuovere l&#39;etanolo svuotando il serbatoio e pompando l&#39;etanolo dal sistema. </li><li> Riempire il tubo da 50 ml di acqua distillata sterile, accendere la pompa e lasciare circolare l&#39;acqua per 30 minuti per sbarazzarsi di etanolo residuo. Rimuovere l&#39;acqua svuotando il serbatoio e pompare l&#39;acqua dal sistema. </li><li> Riempire il tubo da 50 ml con 45 ml di terreno, mettere lo stand nell&#39;incubatrice. Accendere il (velocità di flusso di circa 1 ml / min) e pompa circolare il mezzo di riempire tutti i tubi, rimuovere tutte le bolle d&#39;aria e permettere il mezzo di adattarsi alla composizione del gas nell&#39;incubatrice per almeno 1 ora. Mantenere l&#39;incubatore a condizioni di coltura tissutale-standard (5% di CO 2 e 37 ° C). Assicurarsi che non siano presenti bolle nel tubo. </li></ol> 2. Isolamento di Cuore mouse <ol><li> Iniettare il mouse con 500 unità di eparina. Ciò impedirà la coagulazione del sangue e consente la rimozione di blood dal cuore. Dopo 10 min, anestetizzare il mouse in una camera di induzione con 4% isoflurano utilizzando un vaporizzatore di precisione. L&#39;anestesia è sufficiente quando il mouse non risponde a pizzico piedi. </li><li> Trasferire il mouse per una tavola di dissezione e mantenere l&#39;anestesia mediante una maschera facciale collegato ad un circuito coassiale. Disinfettare la pelliccia del mouse con il 70% di alcol. Utilizzando le forbici aprire la cavità addominale per esporre la vena cava e il diaframma. </li><li> Per esporre il cuore, fare incisioni laterali a partire dall&#39;ultimo al primo costole e riflettono la gabbia toracica sopra la testa di topo. Arrestare il anestesia come mouse non respira più. Osservare il battito del cuore. </li><li> Inserire l&#39;ago 21G attaccato ad una siringa da 5 ml contenente tampone sterile Tyrode nella vena cava inferiore dalla addominale nella cavità toracica (attraverso diaframma). Assicurarsi che il sangue possa uscire dalla vena cava caudale dall&#39;inserzione dell&#39;ago. Profumato °Buffer e Tyrode delicatamente e con pressione costante nella vena cava fino a quando il cuore perde in parte il suo colore rosso. Nota: Il cuore rimane battere permettendo la rimozione del sangue dal cuore. </li><li> Sostituire l&#39;ago con l&#39;ago 21G attaccato ad una siringa da 5 ml contenente soluzione KCl sterile. Profumato delicatamente la soluzione KCl nella vena cava fino a quando il cuore smette di battere e rimuovere l&#39;ago. Nota: La soluzione KCl preserva cuore nella fase diastolica rilassata, che consentirà più facile inserimento degli aghi e perfusione perfusione del sistema coronarico. </li><li> Sollevare leggermente il cuore con pinze curve e con le forbici sezionano il cuore libero dal tessuto circostante, ma lasciano arterie e le vene prossimali al cuore intatto e collegato al cuore. Trasferire il cuore ad una provetta da 15 ml contenente PBS ghiacciato integrato con antibiotici / antimicotici (A / A). Conservare il cuore in PBS freddo per un massimo di 3 ore. </li></ol> 3. Cannulation dei Cuori mouse nella camera di perfusione <ol><li> Eseguire tutte le procedure di cappa a flusso laminare. Trasferire il cuore isolato dal tubo 15 ml per un piatto di 10 centimetri di Petri e aggiungere PBS integrato con A / A. </li><li> Sotto un microscopio da dissezione, utilizzare il micro forbice e pinze per rimuovere tutto il tessuto non cardiaca ma preservare l&#39;aorta ascendente almeno alla biforcazione con l&#39;arteria anonima e le vene polmonari, 2 mm prossimale al cuore. Tagliare la punta della punta del cuore per creare l&#39;accesso al lume del ventricolo sinistro (tipicamente 2 mm). Posizionare la camera di perfusione nella cappa a flusso sotto il microscopio da dissezione. </li><li> Attaccare una siringa da 5 ml con il mezzo all&#39;ago inserto utilizzando il tubo in silicone. Riempire la camera di perfusione con 20 ml di mezzo e mettere il cuore sul palco di rotazione tra i 2 aghi smussati nella camera di perfusione (Figura 1). Regolare l&#39;altezza della fase di rotazione in modo che il cuore è posizionato di fronte allaaghi. </li></ol> 4. legatura per la coltura della valvola mitrale (vedi Figura 1) <ol><li> Legare l&#39;aorta con sutura (seta 7.0). Legare l&#39;appendice atriale sinistra con sutura. Inserire l&#39;ago (nr. 1 in figura 1) attraverso la vena polmonare in atrio sinistro e legare con sutura prossimale alla sua entrata nell&#39;atrio sinistro. Assicurarsi che l&#39;ago non è troppo lontano in atrio sinistro in quanto ciò danneggerebbe la valvola mitrale. </li><li> Inserire l&#39;ago (nr.2 in figura 1) con moto lineare nel ventricolo sinistro. Trasferire il terreno dalla camera di perfusione in una provetta da 50 ml. Sigillare l&#39;ago al miocardio con colla biocompatibile. </li><li> Dopo che la colla si è asciugata, iniettare attentamente medio nel cuore collegando una siringa riempita di medio all&#39;ago nr.1 e verificare se vi siano perdite. Garantire che le uscite medie di ago nr.2 e l&#39;ultimo sangue rimanente viene perfuso dal cuore. </li></ol> 5. Ligazione per la coltura della valvola aortica (vedi Figura 1) <ol><li> Inserire l&#39;ago (nr.1 in figura 1) nell&#39;aorta e legare con suture. Assicurarsi che l&#39;ago non è troppo lontano in aorta poiché ciò danneggiare la valvola aortica. Inserire l&#39;ago (nr.2 in figura 1) con moto lineare nel ventricolo sinistro. Trasferire il terreno dalla camera di perfusione in una provetta da 50 ml. Sigillare l&#39;ago al miocardio con colla biocompatibile. </li><li> Dopo che la colla si è asciugata, iniettare attentamente medio nel cuore collegando una siringa riempita di medio all&#39;ago nr.2 e verificare se vi siano perdite. Garantire che le uscite medie di ago nr.1 e l&#39;ultimo sangue rimanente viene perfuso dal cuore. </li></ol> 6. Mettere Perfusion Camera Stand <ol><li> Riempire camera di perfusione con i 20 ml trasferiti medio. Mettere guarnizione e coperchio sulla camera e stringere con rondella e viti. </li><li> Rimuovere i MTC assemblati dal incubazionetor. Fissare la camera di perfusione sul cavalletto. Collegare il tubo (vedi Figura 1D). Posizionare il supporto in incubatrice (5% di CO 2 e 37 ° C). Collegare il tubo alla pompa. Accendere la pompa con velocità del flusso circa 600 ml / min. </li><li> Assicurarsi che nel serbatoio e / o il gorgogliatore il livello del fluido consente le visualizzazioni della presenza del flusso dalla caduta delle gocce medie in entrata. Dopo coltura, il cuore viene rimosso dal sistema, fissi ed elaborati per l&#39;esame istologico. </li></ol>

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

The authors declare no conflicts of interest.

Fasi critiche coltivando le valvole cardiache di topo comprendono rendendo il tempo tra escissione del cuore dal mouse e la legatura nella camera di perfusione più breve possibile per la sussistenza e legatura degli aghi perpendicolare alle valvole per assicurare la corretta direzione del flusso . Inoltre, il controllo della portata dopo legatura nella camera di perfusione senza prodotto garantisce il corretto inserimento e legatura di aghi. È fondamentale mantenere una cultura sterile, di evitare bolle d&#39;aria nel...

Malattia della valvola cardiaca è una delle principali cause di morbilità e mortalità nel mondo occidentale; la sua prevalenza aumenta con l&#39;età e colpisce oltre il 10% della popolazione di 75 anni e più 1. Le valvole della parte sistemica del cuore, le valvole aortica e mitrale, sono per lo più colpite. Malattia della valvola cardiaca è caratterizzata dalla perdita della struttura altamente organizzata delle valvole, che provoca l&#39;alterazione delle proprietà meccaniche 2. L&#39;integrità strutturale è quindi cruciale per il funzionamento della valvola. Le foglioline della valvola sono costituiti da cellule interstiziali valvolari (VIC), cellule endoteliali valvolari (VEC) e matrice extracellulare, che è altamente organizzato in un modello a strati 3,4. I VIC sono responsabili della sintesi ECM, degradazione e organizzazione. Fattori che emana dal flusso sanguigno, EC o residenti in atto ECM sulle VIC orchestrare la sua funzione. In aggiunta,forze meccaniche del foglio durante il ciclo cardiaco conseguente laminare o sollecitazione di taglio oscillatoria, sollecitazioni di compressione o di trazione che influenzano il comportamento di VIC 5. Per comprendere come è regolata la struttura della valvola, deve prima essere compreso come VIC rispondere alle diverse serie di stimoli sperimentati durante il ciclo cardiaco. Studi in vitro sono stati molto informato circa le caratteristiche e le abilità delle cellule valvolari. La risposta di queste cellule in vitro, tuttavia, non sempre simulare accuratamente la risposta in vivo 6; per esempio, la risposta agli stimoli VIC dipende dalla presenza di cellule endoteliali e la composizione ECM 5. Inoltre, la risposta delle cellule valvolari agli stimoli dipende dalla loro posizione specifica nel foglietto 7. Oltre agli stimoli biochimici, il comportamento delle cellule valvolari è determinata da forze meccaniche o agisconon la valvola 8. Ogni regione della valvola è sottoposto a un insieme specifico di sollecitazioni emodinamiche. Sebbene attuali ex vivo i modelli hanno dimostrato che le forze meccaniche sono importanti determinanti della struttura valvolare 5, i meccanismi associati sono ancora poco chiari. Mentre i modelli in vivo hanno fornito informazioni sui meccanismi molecolari alla base dello sviluppo valvolare 9,10, intuizioni adulto valvolare biologia è ancora sfuggente. Pertanto, un modello in vivo ex flusso è stato sviluppato in cui le valvole cardiache possono essere coltivati ​​nella loro posizione naturale nel cuore per un periodo di tempo prolungato 11. Questo ha il vantaggio che le valvole rimangono nella loro configurazione naturale e le VIC sperimentano lo stesso ambiente in vivo, rendendo le risposte VICS a stimoli più naturale possibile. Inoltre, la cultura delle valvole nella loro posizione naturale nel cuore facilita sottoponendo ogniregione valvolare alle relative sollecitazioni emodinamiche. In questo modello ex vivo, cioè, il Sistema Miniature Tissue Culture (MTC), le valvole possono essere sottoposto a diversi stimoli biochimici ed emodinamici permettendo l&#39;indagine del loro ruolo nel rimodellamento della valvola cardiaca.

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

culturing mouse cardiac valves in the miniature tissue culture system

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

La valvola aortica (Figura 2) o valvola mitrale 11 possono essere coltivate per almeno 3 giorni. Coltivando in posizione aperta (che rappresenta la posizione sistolico della valvola aortica e la posizione diastolica per la valvola mitrale), cellule valvolari rimangono vitali. Nessuna morte cellulare è osservato come determinato dall&#39;assenza di TUNEL-positive cellule (Figura 2H, I) o spaccati caspasi-3 espressione (non mostrato e Lieber et al., 2010 11...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

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

Coltura mouse cardiaca Valvole in miniatura Tissue Culture Sistema

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

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

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JoVE Journal

Bioengineering

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

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

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

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

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

Un metodo per l&#39;incapsulamento follicolo ovarico e la cultura in un proteoliticamente degradabile 3 Sistema dimensionale

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

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Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

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

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

Incapsulamento di cardiomiociti in un idrogel di fibrina per l&#39;ingegneria tissutale cardiaca

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

Capillary Force Lithography for Cardiac Tissue Engineering

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

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

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 ...

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

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

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

Ricostruzioni anatomiche del sistema umano venoso cardiaco utilizzando Contrasto-tomografia computerizzata di campioni di perfusione-fissi

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

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

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

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

Protocollo per la relativa valutazione idrodinamica del Tri-volantino Polymer Valvole

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

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

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

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

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

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

Un<em&gt; In Vitro</em&gt; Organ Culture Modello del disco intervertebrale Murine

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

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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

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

Coltura di cellule di mammifero in scaffold tridimensionale Peptide

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

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

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

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

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. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

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

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

Caratterizzazioni biassiale meccaniche delle valvole atrioventricolari cuore

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