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.

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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.

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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 ...

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18.9K 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

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Manipolazione delle cellule viventi per costruire assieme cellulare 3D stabile senza impalcatura artificiale

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Ricerca

Biochimica

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

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

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

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

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

Bioingegneria

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. ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Tecnica Automated robotica erogazione di orientamento della superficie e Bioprinting di celle

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Scaling di Engineered vascolari innesti Usando 3D Stampato Guide e il metodo di impilamento Anello

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularized microfluidic multimateriali per l'ingegneria dei tessuti e Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabbricazione di pozzi di agarosio personalizzato per la semina delle cellule e tessuto anello auto-assemblaggio utilizzando stampi 3D-stampato

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Modellazione semplice, economica e modulare delle cellule utilizzando il DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Diretto Cellular Self-Assembly di fabbricare Cell-Derived Anelli tessuto per l'analisi biomeccanica e di ingegneria dei tessuti

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