Gli autori ringraziano Neil Whitehouse (WPI, Machine Shop Higgins) per la sua assistenza con lavorazione CNC. Inoltre, vorremmo ringraziare Adriana Hera (WPI Computing and Communications Center) per il suo aiuto con MATLAB di programmazione, così come Kate bevande e Giuseppe Cotnoir (WPI Academic Technology Center) per l&#39;assistenza con Camtasia. Sophie Burke e Jacleen Becker (WPI Academic Technology Center), a condizione supplementare filmati video. Questo lavoro è stato finanziato dal National Institutes of Health (R15 HL097332), il Massachusetts Medical School-WPI Research Initiative pilota, l&#39;American Heart Association (borsa di studio di laurea a JZH), e Worcester Polytechnic Institute (Undergraduate Research Fellowship Estate a JZH e istituzionali start-up fondi per MWR).

1. Cellula semina fabbricazione dello stampo Iniziare mediante fresatura da un 1 / 2 &quot;pezzo spesso di policarbonato per creare 15, a fondo tondo, pozzi anulare con diametro del perno di centraggio di 2 mm. I canali fresato sono 6 mm di profondità e 3,75 millimetri di larghezza. Pulire e asciugare lo stampo in policarbonato per rimuovere eventuali residui di plastica dal processo di fresatura. Mix polidimetilsilossano (PDMS) con un rapporto 10:1 (w / w) di base a catalizzatore, Degas per rimuovere tutte le bolle d&#39;aria, e versare sopra lo stampo in policarbonato. Degas di nuovo per rimuovere eventuali bolle rimanenti, e la cura in forno a 60 ° C per 4 ore. Una volta indurito, rimuovere con attenzione il modello da PDMS lentamente peeling dal policarbonato, lavare con acqua e sapone, e autoclave. Anche autoclave una soluzione di agarosio per cento due (w / v) disciolto in Dulbecco modificato (DMEM). Posizionare il modello PDMS su una superficie piana e riempire con fuso aga<html> è aumentato del primo pipettaggio agarosio in ciascuno dei stampi posta al centro, e poi pipettaggio nello spazio intorno ad esso. Lasciare solidificare l&#39;agarosio (circa 15 minuti), poi capovolgere lo stampo per liberare l&#39;agarosio dal PDMS. Tagliare l&#39;eccesso di agarosio di tutto ognuno dei pozzi, i pozzi e il luogo di agarosio in 12-pozzetti. 2. Coltura cellulare e semina anello Aggiungi coltura cellulare media (DMEM con 10% FBS e 1% penicillina-streptomicina) intorno alla parte esterna dello stampo agarosio, senza coprire la parte superiore degli stampi. Posizionare le piastre nell&#39;incubatore e permettere loro di equilibrare per circa 15 minuti mentre preparate le cellule. Al 90% confluenza, trypsinize topo cellule muscolari lisce aortiche (rSMCs) e risospendere ad una concentrazione di 5x10 6 cellule / ml. Pipettare 100 l di sospensione cellulare in ciascuno dei anulare agarosio pozzi, con un movimento circolare per applicare le cellule in ogni pozzetto. contenuto &quot;&gt; Assicurarsi che gli scaffali sono incubatore livello, quindi posizionare le piastre nell&#39;incubatrice e permettere loro di riposare per 24 ore. medium Cambio ogni due giorni successivi aspirando i media di tutto il bene, e ri-riempire ogni bene fino alla agarosio bene è completamente sommerso. 3. Tessuto raccolta anello e misure di spessore A conclusione della cultura, rimuovere l&#39;anello dallo stampo agarosio facendolo scorrere sopra la parte superiore del montante centrale. Posizionare l&#39;anello in una piccola scatola di Petri con tampone fosfato (PBS), centro del campione, e acquisire un&#39;immagine utilizzando un sistema di imaging digitale. Utilizzare un programma di analisi delle immagini per il rilevamento dei bordi e misurare lo spessore dell&#39;anello in 4 posizioni (alto, basso, sinistra e destra) lungo la sua circonferenza. Calcolare il valore medio spessore (t) per ogni singolo anello, e usare questo valore per calcolare la sezione trasversale(2π (t / 2) 2). 4. Prove meccaniche di anelli Impostare la macchina di prova a trazione (Instron EPS 1000) in posizione orizzontale. Collegare un 1N ± 1mn cella di carico e personalizzati impugnature filo sottile (creato da piegare filo di acciaio inossidabile). Montare l&#39;anello di campione sulle due impugnature filo sottile. Estendere le prese fino a 5 mN carico tara è applicata al campione. Inserire la sezione trasversale (in base a misurazioni di spessore) e registrare il tratto utile. Pre-ciclo gli anelli 8 volte tra il carico di stress tara e 50 kPa ad una velocità di 10 mm / min. Dopo l&#39;ottavo pre-ciclo, tirare al fallimento a 10 mm / min. Il fallimento è notato come una diminuzione della forza del 40% da una misura all&#39;altra. Per ogni campione anello, misurare lo sforzo finale trazione (UTS) e tensioni fallimento e calcolare il massimo tangente modulo (MTM) a partire dai dati acquisiti. 5. Tessuto anellotaggio per fabbricare tubi di tessuto Portaprovette personalizzati sono lavorati da rotondo 5 centimetri dischi di policarbonato con ritagli rettangolari (2 cm x 3 cm). Praticare i fori filettati con questi dischi per consentire a due titolari di essere avvitati insieme. Autoclave i titolari tubo personalizzato e quindi il trasferimento al mobile biosicurezza e metterlo in un piatto vuoto Petri. Usare le forbici chirurgiche per tagliare le estremità dei tubi di 1,9 mm di diametro in silicone con un angolo per creare bordi smussati, e poi in autoclave i tubi in silicone. Mentre i tubi sono stati sterilizzati in autoclave, riempire una piastra di Petri con i media. Rimuovere gli anelli dai pozzi agarosio e metterli nella scatola di Petri con i media. Luogo in autoclave i tubi di silicone nello stesso piatto di Petri per bagnare prima dell&#39;uso. Utilizzare pinze per mettere un fine smussata di un tubo di silicone al centro di un anello e far scorrere l&#39;anello sul tubo in silicone. Ripetere con il numero desiderato di riNGS. Far scorrere gli anelli in contatto uno con l&#39;altro da delicatamente spingendo successivamente in entrambe le direzioni lungo il tubo. Questo metodo può essere utilizzato per gli anelli raccolti dopo un solo giorno in coltura. Una volta che gli anelli sono montati, allineare i tubi di silicone all&#39;interno della titolari policarbonato e avvitare le due parti del titolare insieme. Posizionare il supporto in un piatto di 100 mm di Petri e aggiungere 55 ml multimediali. Scambio dei media ogni 3 giorni per la durata della cultura. Per rimuovere i tubi di tessuto, rilasciare i tubi in silicone dal supporto in policarbonato e utilizzare pinze per far scorrere i tubi di tessuto dal tubo in silicone e in una piastra di Petri riempite con PBS. 6. Rappresentante dei risultati: Quando il protocollo è eseguita correttamente, le cellule si aggregano formando anelli di tessuto con un diametro interno uguale al diametro del perno stampo corrispondente entro 24 ore. I bordi lisci ad anello sono di solito in apparenza e (se cultured su una superficie piana), sono di spessore uniforme lungo l&#39;intero perimetro. Gli anelli di tessuto sono facili da gestire e possono essere rimossi dai loro pozzi per le successive analisi meccaniche e istologiche (v., in Gwyther et al. 2011, 8). Anello morfologia del tessuto, la composizione della matrice, e le proprietà meccaniche variano a seconda del numero e del tipo di cellule inseminate. Risultati rappresentativi da anelli di tessuto a base di diversi tipi di cellule, semina le condizioni, e la lunghezza della cultura sono riportati nella tabella 1. Due anelli mm ID creato da 5x10 5 rSMCs esibito un carico di rottura superiore a quello anelli creati dalla nostra precedentemente pubblicato con 2 anelli mm (fatto da 6.6x10 5 rSMCs). Simili alle cellule di ratto, cellule muscolari lisce (HSMC) 8 facilmente aggregate per formare il tessuto Anelli, che conteneva una grande quantità di collagene dopo soli 14 giorni in coltura (dati non riportati). Cellule staminali mesenchimali (hMSC) anche aggregati e formano anelli coesa, ma lacked resistenza meccanica e si è rotto durante le fasi iniziali di precycling prove di trazione uniassiale. <table border="1"><tbody><tr><td></td><td> n </td><td> Numero di cellule </td><td> Spessore (mm) </td><td> Lunghezza della cultura (giorni) </td><td> UTS (kPa) </td><td> MTM (kPa) </td><td> Il mancato ceppo (mm / mm) </td></tr><tr><td> rSMC anelli </td><td> 6 </td><td> 660.000 </td><td> 0,94 ± 0,12 </td><td> 14 </td><td> 97 ± 30 </td><td> 497 ± 91 </td><td> 0,50 ± 0,08 </td></tr><tr><td> rSMC anelli </td><td> 4 </td><td> 500.000 </td><td> 0,53 ± 0,02 </td><td> 7 </td><td> 113 ± 8 </td><td> 189 ± 15 </td><td> 0,88 ± 0,05 </td></tr><tr><td> HSMC anelli </td><td> 3 </td><td> 750.000 </td><td> 0,51 ± 0,05 </td><td> 14 </td><td> 160 ± 30 </td><td> 270 ± 20 </td><td> 0,92 ± 0,08 </td></tr><tr><td> hMSC anelli </td><td> 3 </td><td> 750.000 </td><td> 0,40 ± 0,07 </td><td> 14 </td><td> N / A </td><td> N / A </td><td> N / A </td></tr></tbody></table> Tabella 1. Tabella che mostra i parametri della cultura e delle proprietà meccaniche dei 2 anelli millimetri generate da diversi tipi di cellule, le concentrazioni di semina, e durate la cultura. <img alt="Figura 1" src="/files/ftp_upload/3366/3366fig1.jpg" /> Figura 1. (A) Schema del processo di generazione dei tessuti anello. (B) Personalizzato stampo in policarbonato con fresata pozzi anulare. Diametri postale centrale sono 2 mm. (C) PDMS modello dopo che è stato staccato dallo stampo in policarbonato. (D) anello di tessuto aggregati in coltura in uno stampo agarosio con un post 2 mm. (E) Due millimetri di diametro anello di tessuto in PBS. Barre di scala = 6 mm (B, C) e 2 mm (D, E). <img alt="Figura 2" src="/files/ftp_upload/3366/3366fig2.jpg" /> Figura 2. Trama rappresentante della curva produttori sforzo-deformazioneced da prove di trazione uniassiale.

<ol>
	<li>Roger, V. L. <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--2011+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2011 Update: A Report From the American Heart Association.</a> Circulation. 123, e18-e209 (2011).</li><li>Kelm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+concept+for+scaffold-free+vessel+tissue+engineering:+self-assembly+of+microtissue+building+blocks.">A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.</a> J. Biotechnol. 148, 46-55 (2010).</li><li>Gauvin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+single-step+self-assembly+approach+for+the+fabrication+of+tissue-engineered+vascular+constructs.">A novel single-step self-assembly approach for the fabrication of tissue-engineered vascular constructs.</a> Tissue. Eng. Part. A. 16, 1737-1747 (2010).</li><li>L'Heureux, N., McAllister, T. N., de la Fuente, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+blood+vessel+for+adult+arterial+revascularization.">Tissue-engineered blood vessel for adult arterial revascularization.</a> N. Engl. J. Med. 357, 1451-1453 (2007).</li><li>Norotte, C., Marga, F. S., Niklason, L. E., Forgacs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+vascular+tissue+engineering+using+bioprinting.">Scaffold-free vascular tissue engineering using bioprinting.</a> Biomaterials. 30, 5910-5917 (2009).</li><li>Dean, D. M., Napolitano, A. P., Youssef, J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rods,+tori,+and+honeycombs:+the+directed+self-assembly+of+microtissues+with+prescribed+microscale+geometries.">Rods, tori, and honeycombs: the directed self-assembly of microtissues with prescribed microscale geometries.</a> FASEB. J. 21, 4005-4012 (2007).</li><li>Livoti, C. M., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+and+tissue+fusion+of+toroid-shaped+minimal+building+units.">Self-assembly and tissue fusion of toroid-shaped minimal building units.</a> Tissue. Eng. Part. A. 16, 2051-2061 (2010).</li><li>Gwyther, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+vascular+tissue+fabricated+from+aggregated+smooth+muscle+cells.">Engineered vascular tissue fabricated from aggregated smooth muscle cells.</a> Cell. Tissues. Organs. 194, 13-24 (2011).</li><li>Seliktar, D., Black, R. A., Vito, R. P., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+mechanical+conditioning+of+collagen-gel+blood+vessel+constructs+induces+remodeling+in+vitro.">Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro.</a> Ann. Biomed. Eng. 28, 351-362 (2000).</li><li>Rowe, S. L., Stegemann, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpenetrating+collagen-fibrin+composite+matrices+with+varying+protein+contents+and+ratios.">Interpenetrating collagen-fibrin composite matrices with varying protein contents and ratios.</a> Biomacromolecules. 7, 2942-2948 (2006).</li><li>Pins, G. D., Collins-Pavao, M. E., De Water, L. V. a. n., Yarmush, M. L., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+triggers+rapid+contraction+and+degradation+of+fibroblast-populated+collagen+lattices.">Plasmin triggers rapid contraction and degradation of fibroblast-populated collagen lattices.</a> J. Invest. Dermatol. 114, 647-653 (2000).</li><li>Konig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+completely+autologous+human+tissue+engineered+blood+vessels+compared+to+human+saphenous+vein+and+mammary+artery.">Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.</a> Biomaterials. 30, 1542-1550 (2009).</li><li>Mironov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+tissue+spheroids+as+building+blocks.">Organ printing: tissue spheroids as building blocks.</a> Biomaterials. 30, 2164-2174 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Recentemente, c&#39;è stato un crescente interesse a base di cellule o &quot;patibolo-less&quot; metodi di ingegneria tissutale per affrontare alcuni dei limiti degli approcci tessuto ponteggio a base di ingegneria. Dato che derivati ​​dalle cellule dei tessuti vengono create dalle cellule e la matrice che producono, che contengono intrinsecamente molto più alta densità di cellulari, non contengono materiali esogeni, e può essere realizzato interamente in cellule umane e proteine. Innesti vascolari costituito da cellule umane possono raggiungere notevoli resistenze meccaniche in assenza di ponteggi esogeni (per esempio, pressioni di scoppio 3400 mmHg rispetto a 1600 mmHg per l&#39;uomo safene) 12. Anche se i tessuti vascolari a base di cellule mostrano densità cellulare migliorata e resistenza meccanica, metodi di fabbricazione più recente (come &quot;foglio-based engineering&quot; 3,4,12 o &quot;bioprinting&quot; 5,13) richiedono periodi di cultura estesa (&gt; 3 mesi) o attrezzature specializzate per la costruzione del tessuto 3D. I derivati ​​dalle cellule del tessuto anello method qui descritto consente una rapida cellulari auto-assemblaggio a formare solide costruzioni tessuto 3D in un breve periodo di tempo e senza l&#39;uso di attrezzature specializzate. Questo protocollo in dettaglio la procedura che abbiamo sviluppato per creare 2 millimetri di diametro interno ratto liscia derivati ​​dalle cellule del tessuto muscolare anelli. Nel presente esempio, anelli di tessuto sono stati coltivati ​​per 7 giorni (poi coltivate per altri 7 giorni per la fusione anello e la formazione del tubo). Tuttavia, 2 ratti mm (e umano) delle cellule muscolari lisce anelli possono essere rimosse dai pozzi e sono abbastanza coesa per la gestione (ad esempio, il trasferimento su tubi in silicone) già un giorno dopo la semina delle cellule. Inoltre, gli anelli di tessuto robusto con diversi diametri interni (2, 4 e 6 mm) possono essere creati con questo metodo semplicemente cambiando il diametro posto di stampo originale in policarbonato. 8 Abbiamo anche recentemente modificato la progettazione dello stampo in policarbonato per consentire cinque 2 millimetri pozzi semina per essere gettati in un unico multi-bene agarosio camera, cheutilizza meno PDMS e agarosio, e si inserisce in un pozzetto di un 6-pozzetti (dati non riportati). Le variazioni di diametro posta, la larghezza della semina bene, il raggio di curvatura del fondo arrotondato, il numero di pozzetti semina, o la profondità dei pozzi di semina possono essere modificati semplicemente cambiando le specifiche del file CAD per CNC lavorazione dello stampo in policarbonato. Infine, un unico stampo in policarbonato può essere usato per fabbricare un numero illimitato di modelli PDMS, PDMS e ogni template può essere pulito, autoclave e riutilizzati decine di volte. Oltre a modificare la dimensione degli anelli di tessuto, abbiamo fatto gli anelli da molti tipi di cellule diverse, tra cui: SMC primari di ratto (Applicazioni Cell, R354-05), primaria SMC umano coronarica (Lonza, CC-2583), umano primario fibroblasti dermici 11 (dono generoso del dottor George Pins, WPI Dipartimento di Ingegneria Biomedica), fibroblasti di polmone di ratto (RFL-6, ATCC CCL-192), e le cellule staminali mesenchimali (Lonza, PT-2501). Ognuno di questi tipi di aggregati cellulari e contratti di tutto il centro messaggi per formare anelli di tessuto, anche se l&#39;organizzazione cellulare, composizione ECM, e le proprietà meccaniche dei costrutti variano per ogni tipo di cellula. I parametri di semina per ciascun tipo di cella deve essere empiricamente determinato sulla base della dimensione delle celle e la loro capacità di aggregare. Pertanto, mentre questo sistema di creazione di cellule derivate anelli tessuto è estremamente versatile, il protocollo può avere bisogno di piccoli aggiustamenti formazione di tessuto ottimale con diversi tipi di cellule. Anello geometria tessuto facilita il caricamento facile e valutazione delle proprietà dei materiali tessuti da prove di trazione uniassiale, come descritto. Ci sono anche precedenti sostanziale per l&#39;utilizzo di segmenti di anello di vasi sanguigni per misurare la contrazione vascolare e funzione fisiologica. Studi preliminari indicano che derivati ​​dalle cellule anelli di tessuto può essere montato su un dispositivo miografo fili per la misura di risposta farmacologica eforza contrattile generazione (dati non riportati). Complessivamente, la capacità di fabbricare rapidamente auto-assemblati anelli di cellule per l&#39;analisi istologica, meccanici, fisiologici, biochimici e suggerisce un nuovo e potente strumento che può essere utile per modellare la struttura del tessuto vascolare e la funzione nella salute e nella malattia.

<table border="1"><tbody><tr><th> Nome del reagente / apparecchiatura </th><th> Azienda </th><th> Catalogo </th></tr><tr><td> Polidimetilsilossano (PDMS) </td><td> Dow Corning </td><td> Sylgard 184 </td></tr><tr><td> Agarosio </td><td> Lonza </td><td> 50000 </td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 15-017-CV </td></tr><tr><td> Siero fetale bovino (FBS) </td><td> PAA </td><td> A05-201 </td></tr><tr><td> Penicillina / streptomicina </td><td> Mediatech </td><td> 30-002-CI </td></tr><tr><td> Sistema di imaging digitale </td><td> DVT Corporation </td><td> Modello 630 </td></tr><tr><td> Collaudo della macchina monoassiale </td><td> Instron </td><td> ElectroPuls E1000 </td></tr><tr><td> Rilevamento dei bordi Software </td><td> DVT Corporation </td><td> Framework 2.4.6 </td></tr></tbody></table>

directed cellular self-assembly to fabricate cell-derived tissue rings for biomechanical analysis and tissue engineering

Ogni anno, centinaia di migliaia di pazienti sottoposti ad intervento di bypass coronarico negli Stati Uniti 1. Circa un terzo di questi pazienti non hanno vasi adatti donatore autologo a causa della progressione della malattia o raccolto precedente. Lo scopo di ingegneria dei tessuti vascolari è quello di sviluppare una fonte alternativa adatta per questi innesti di bypass. Inoltre, il tessuto vascolare progettato può risultare prezioso come modelli viventi vascolare per lo studio delle malattie cardiovascolari. Diversi approcci promettenti ai vasi sanguigni di ingegneria sono state esplorate, con molti studi recenti incentrati sullo sviluppo e l&#39;analisi di metodi basati su celle. 2-5 Qui, presentiamo un metodo rapido per auto-assemblano in anelli cellule del tessuto 3D che possono essere utilizzati in vitro per modellare i tessuti vascolari. Per fare questo, sospensioni di cellule muscolari lisce sono seminate in fondo rotondo anulare agarosio pozzi. La non-adesivo proprietà del agarosio permettono °cellule e per risolvere, aggregare e contratto intorno ad un perno posto al centro del pozzo per formare un anello coeso tessuto. 6,7 Questi anelli possono essere coltivate per diversi giorni prima della raccolta per i meccanici, fisiologica, l&#39;analisi biochimica, o istologici. Abbiamo dimostrato che questi derivati ​​dalle cellule del tessuto anelli rendimento a 100-500 la resistenza alla trazione kPa ultimo 8, che supera il valore riportato per altri costrutti vascolari di ingegneria tessutale in coltura per eguali periodi (&lt;30 kPa). 9,10 I nostri risultati dimostrano che la cellula robusta di derivazione vascolare generazione anello di tessuto può essere raggiunto entro un breve periodo di tempo, e offre l&#39;opportunità di valutazione diretta e quantitativa dei contributi di cellule e derivati ​​dalle cellule della matrice (CDM) di struttura del tessuto vascolare e funzione.

Watch this Scientific Journal Video about Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering at JoVE.com

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Questo articolo descrive un metodo versatile per creare cellule derivate da anelli di tessuto cellulare self-assembly. Cellule muscolari lisce seminate in forma di anello aggregato agarosio pozzi e contratto a formare solide tridimensionali (3D) nei tessuti entro 7 giorni. Millimetrica scala anelli di tessuto sono favorevoli ai test meccanici e servono come mattoni per l&#39;assemblaggio dei tessuti.

Diretto Cellular Self-Assembly di fabbricare Cell-Derived Anelli tessuto per l&#39;analisi biomeccanica e di ingegneria dei tessuti

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

directed-cellular-self-assembly-to-fabricate-cell-derived-tissue

Research

JoVE Journal

Bioengineering

18.7K Views.  Worcester Polytechnic Institute. Questo articolo descrive un metodo versatile per creare cellule derivate da anelli di tessuto cellulare self-assembly. Cellule muscolari lisce seminate in forma di anello aggregato agarosio pozzi e contratto a formare solide tridimensionali (3D) nei tessuti entro 7 giorni. Millimetrica scala anelli di tessuto sono favorevoli ai test meccanici e servono come mattoni per l'assemblaggio dei tessuti.

Video: Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

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

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

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

Ricerca

Bioingegneria

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Di campo elettrico controllato migrazione diretta di cellule progenitrici neurali in ambienti 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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

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

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

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

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

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

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

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

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

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

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

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Sintesi di cationizzata Magnetoferritin per la magnetizzazione ultra-veloce di celle

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...