Il finanziamento è stato fornito dal National Institutes of Health (EB 001013), scienze naturali e ingegneria Research Council del Canada e Canadian Institutes of Health Research. Ringraziamo il Dott. AP McGuigan per la sua esperienza e aiutare nello sviluppo di moduli.

1) Preparazione dei tubi <ol><li> Tagliare 2-3 m di lunghezza di tubo in polietilene (0,76 mm ID x 1,22 mm di diametro). </li><li> Infilare un ago 20 G1 in una delle estremità del tubo. </li><li> Bobina il tubo e posto in un self-sigillo sacchetto convertitori (7 1 / 2 &quot;x 13&quot;). Le due estremità del tubo deve essere collocato alla fine non sigillata della borsa e registrato sul posto con nastro a gas sterilizzazione. </li><li> Borsa di tenuta e di gas sterilizzare. </li></ol> 2) Neutralizzazione di Collagene Nota: 1 ml di collagene riempie circa 2 metri di tubo in polietilene. <ol><li> Mettere la quantità necessaria di collagene necessario per la lunghezza desiderata del tubo in un tubo da 15 ml e continuare su ghiaccio. </li><li> Aggiungere 128 uL di 10x α-MEM per mL di collagene per il tubo e mescolare pipettare ripetutamente la soluzione. Nota: (1) La soluzione dovrebbe diventare giallo come la α-MEM è mescolato nel collagene acidificato e (2) cercare di minimizzare le bolle in fase di missaggio. </li><li> Neutralizzare il collagene con l&#39;aggiunta di bicarbonato di sodio 0,8 M fino a quando il collagene, ha un pH di 7,4. Per una stima generale del bicarbonato di sodio pH aggiungere fino a quando la soluzione di collagene giallo diventa di colore rosa chiaro. Nota: Si richiede in genere 30-60 microlitri per ogni ml di collagene. </li><li> Tenere collagene neutralizzato sul ghiaccio fino al momento dell&#39;uso, come il collagene viene neutralizzata gel se non mantenuto a 4 ° C. </li></ol> 3) Integrazione delle celle (opzionale) Nota: a seconda del tipo di cellula le cellule possono essere incorporati ad una concentrazione compresa tra 1 milione di cellule per ml a 20 milioni di cellule per ml. Utilizzare il supporto necessario per le celle che si desidera incorporare. <ol><li> Rimuovere i supporti dalle cellule e lavare con 5 ml di PBS. </li><li> Rimuovere PBS e aggiungere 3 mL tripsina-EDTA. </li><li> Incubare le cellule in tripsina-EDTA per 5 min a 37 ° C. </li><li> Risospendere le cellule in 7 dei media ml. </li><li> Contare le celle. </li><li> Trasferire il numero necessario di cellule per l&#39;incorporamento di un tubo da 15 ml. </li><li> Agglomerare le cellule a 300 xg per 5 min. </li><li> Aspirare i media fuori il pellet cellulare. </li><li> Risospendere le cellule del collagene neutralizzato e tenere in ghiaccio. </li></ol> 4) gelificanti di Collagene <ol><li> In una cappa di sicurezza biologica tagliare la fine della busta sigillata contenente il tubo in polietilene. </li><li> Allegare una siringa da 3 ml l&#39;ago 20G, alla fine, uno dei tubi con delle pinzette sterilizzate. Tenere il tubo nella borsa. </li><li> Inserire l&#39;altra estremità del tubo nel collagene neutralizzato fino a quando la punta si trova al fondo della provetta conica con una pinzetta sterilizzata. Tenere il tubo conico sul ghiaccio. </li><li> Tirare il collagene nel tubo in polietilene da ritraendo lo stantuffo della siringa lentamente. Nota: Se il collagene è tirato nel tubo per poi rapidamente bolle si formano nel collagene. </li><li> Dopo aver estratto il collagene attraverso il tubo, estrarre l&#39;ago 20G di tubi e mettere entrambe le estremità indietro nel sacco. Nastro la borsa chiusa. </li><li> Incubare il sacchetto a 37 ° C per 60 min. Se il collagene contiene cellule poi capovolgere il sacchetto sopra ogni 15 min. in modo che le cellule non accontentarsi di una parte del modulo. </li></ol> 5) Taglio di tubi contenenti il ​​collagene gelificati <ol><li> Intestazioni autoclave e la lama per il dispositivo di taglio e il vassoio per contenere tubo. </li><li> Montare il dispositivo di taglio. </li><li> Tubo posto in un vassoio sterile di fronte a taglio dispositivo e caricare il tubo nel dispositivo di taglio. </li><li> Impostare un tubo da 50 ml conica contenente 25 mL di mezzi in uscita del dispositivo di taglio per raccogliere i moduli di taglio. Nota: i media Usa contenente FBS, anche per il collagene, solo i moduli come i moduli non separato dal tubo senza FBS nei media. Se i moduli contengono cellule utilizzano il supporto richiesto per le cellule embedded. Se non ci sono cellule incorporati nei moduli di usare i media per le cellule endoteliali. </li><li> Impostare il dispositivo di taglio per taglio 2 mm di lunghezza di tubo e tagliare il tubo. </li><li> Incubare tagliare i moduli a 37 ° C per 60 min nel tubo da 50 ml. </li></ol> 6) Rimozione dei moduli collagene da tubi <ol><li> Vortex tubo 50 ml conica contenente moduli nel tubo per 10 secondi. Nota: Sii gentile quando separazione moduli integrati con celle ad alta densità per evitare la rottura. </li><li> Lasciate che i moduli depositano sul fondo della provetta. Dura circa 5 minuti. </li><li> Utilizzando un ampio bocca pipetta sierologica 10 ml trasferire i moduli si stabilirono in una provetta da 15 ml. </li><li> Ripetere i passaggi 6,1-6,3 due volte. </li><li> Passare al punto 7 per moduli cappotto con le cellule endoteliali o moduli di trasferimento a un non-tessuto piastra di coltura trattata. </li></ol> 7) rivestimento dei moduli con le cellule endoteliali <ol><li> Risolvere i moduli sul fondo di un tubo da 15 ml coniche con 5 ml di media per °e incorporati cellule. </li><li> Rimuovere i supporti dalle cellule endoteliali e lavare con 5 ml di PBS. </li><li> Rimuovere PBS e aggiungere 3 mL tripsina-EDTA. </li><li> Incubare le cellule in tripsina-EDTA per 5 min a 37 ° C. </li><li> Risospendere le cellule in 7 dei media ml. </li><li> Contare le celle. Hai bisogno di 5x10 6 cellule endoteliali per ml di moduli imballato nella parte inferiore del tubo conico. </li><li> Agglomerare le cellule a 300 xg per 5 minuti e risospendere in 5 ml di mezzi di comunicazione delle cellule endoteliali. </li><li> Aggiungere i 5 ml di supporti contenenti cellule endoteliali ai moduli. Nota: Questo dà un rapporto di 50:50 dei due tipi di supporto che abbiamo trovato a lavorare per la sopravvivenza e la funzione dei due tipi di cellule. Testare l&#39;utilizzo di co-cultura medio formulato per garantire una corretta manutenzione dei fenotipi dei due tipi di cellule prima di utilizzarlo. </li><li> Incubare i moduli con le cellule endoteliali sia staticamente e dinamicamente. </li><li> Per i moduli statici mix di incubazione con cellule endoteliali per inversione e la camera d&#39;aria in posizione verticale a 37 ° C per 15 min. Ripeti. </li><li> Dinamicamente seme le cellule endoteliali a dondolo con delicatezza il tubo a 37 ° C per 60 min. </li><li> Ripetere l&#39;incubazione statica due volte. </li><li> Riposizionare i moduli in un piatto non tessuto coltura trattata e di incubare una notte a 37 ° C. </li><li> Il luogo giorno dopo, i moduli in una nuova non-tessuto piastra di coltura trattata con mezzi freschi (miscela 50:50 se si usa co-coltura di sistema) per rimuovere unattached cellule endoteliali. </li><li> Incubare i moduli che sono rivestite con le cellule endoteliali fino a che non coprono il 100% della superficie dei moduli. Questo richiede di solito circa 7 giorni. </li></ol> 8) Risultati Rappresentante: I moduli cilindrici sarà la prima volta che vengono rimossi dal tubo. Se i moduli contengono cellule embedded e / o sono rivestite di cellule endoteliali possono contratto fino al 50% in volume e sviluppare una forma ovale (Figura 1). I moduli diventerà anche più denso e più opaco se visti al microscopio ottico (figura 1). Anche quando le cellule endoteliali sono confluenti sulla superficie dei moduli vi è una formazione di giunzioni strette, che può essere visto da immunofluorescenza di VE-caderina (Figura 2). Trasferimento di massa analisi ha dimostrato che i moduli sono in grado di supportare celle ad alta densità (8x10 7 cellule / cm 3) senza lo sviluppo di un core necrotico a causa di trasportare l&#39;ossigeno inadeguato, che è spesso problematico in più tessuti (ad esempio moduli di grande diametro, D = 1,4 mm) (Figura 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Figura 1" /> Figura 1: Modulo di fabbricazione e contrazione. Contrazione del modulo si sono verificati durante i tre giorni successivi HUVEC-C (linea di cellule endoteliali) semina risultati significativamente più piccola di diametro modulo e la lunghezza (p &lt;0,001) (A). Incorporato cellule HepG2 sono stati uniformemente distribuiti all&#39;interno di moduli al momento della fabbricazione e mantenuto alta la vitalità (B). [Live cellule verdi, le cellule morte red] [adattato da Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Figura 2" /> Figura 2: VE-caderina colorazione di immunofluorescenza di giunzioni cellulari CE strette 10 giorni dopo RAEC dove rivestito sulla superficie di un modulo. Barra di scala = 50 micron. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Figura 3" /> Figura 3: colorazione tricromica di Masson di moduli tipici (0,76 mm di diametro iniziale) e moduli di grandi dimensioni (1,4 mm di diametro iniziale) dimostrare l&#39;effetto delle limitazioni di diffusione dell&#39;ossigeno. Sette giorni dopo la fabbricazione, un gran numero di cellule morte si era formata all&#39;interno del nucleo dei moduli di grandi dimensioni (pannello in basso a destra), lasciando solo un bordo sottile (~ 200μm di spessore) di cellule vitali. Al contrario, i piccoli moduli mantenuto una distribuzione uniforme ed elevato di cellule vive. [Moduli embedded con cellule HepG2 e rivestite con le cellule endoteliali.] [Tratto da Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Figura 4" /> Figura 4: (A) HMEC-1 seminati su poloxamine - moduli di collagene. Dopo Giorno 1 del seeding del poloxamine-collagene moduli mantengono la loro forma cilindrica e non vi è limitata proprietà attaccamento delle cellule rispetto ai moduli collagene solo. Barra di scala = 200 micron. (B) Immagine al microscopio a luce di un modulo di collagene contenenti microsfere a base di PLGA biodegradabile. Barra di scala = 500 micron. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Figura 5" /> Figura 5: Esempi di test in vitro. (A) saggio Angiogenesi: capillare come formazioni sul modulo seminati con cellule endoteliali possono essere facilmente individuati e quantificati dopo 5 giorni di incubazione con derivate da tessuto adiposo cellule staminali. (B) immagini di microscopia confocale di un saggio dal vivo / morto sui moduli in cui la snenti di cellule vive è verde e per le cellule morte è rosso. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Figura 6" /> Figura 6: i moduli di collagene contenenti incorporato cellule staminali mesenchimali e di uno strato superficiale di cellule endoteliali. I moduli sono stati esposti a sollecitazioni di taglio (~ 0,64 dyn / cm 2) per 7 giorni in una camera di microfluidica e cominciano a mostrare la fusione nei punti di contatto. Le cellule endoteliali sono colorati con vWF (marrone) e nuclei di cellule staminali mesenchimali appaiono blu (H &amp; E). Barra di scala = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Figura 7" /> Figura 7: Centinaia di moduli di collagene contenenti derivate da tessuto adiposo cellule staminali (ASC) e rivestito con cellule endoteliali microvascolari (HMEC) vengono impiantate sotto la pelle del topo per studiare ASC / HMEC interazione in vivo per le applicazioni di rigenerazione grasso.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. 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+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. 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+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo fabbricato diversi diversi micro-tessuti con moduli integrati con diversi tipi di cellule 1-11. Abbiamo incorporato con successo cardiomiociti primari, isolotti, le cellule staminali adipose e cellule stromali mesenchimali così come diverse linee cellulari tra cui HepG2, NIH 3T3 e clonare 9 cellule del fegato. Abbiamo ricoperto i moduli con vari tipi di cellule endoteliali, comprese le cellule endoteliali aortiche ratto, cellule endoteliali della vena ombelicale e le cellule endoteliali microvascolar...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
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fabrication of micro-tissues using modules of collagen gel containing cells

Questo protocollo descrive la fabbricazione di un tipo di micro-tessuti chiamate moduli. L&#39;approccio modulo genera uniforme, scalabile e tessuti vascolarizzati. I moduli possono essere fatte di collagene così come altri materiali gelable o reticolabile. Sono circa 2 mm di lunghezza e 0,7 mm di diametro sulla fabbricazione, ma ridursi in dimensione con le cellule incorporato o quando i moduli sono rivestite di cellule endoteliali. I moduli singolarmente sono abbastanza piccoli che le celle sono incorporate nel limitare la diffusione di ossigeno e altre sostanze nutritive, ma i moduli possono essere imballate insieme per formare tessuti che sono più grandi perfusable. Questi tessuti sono modulari in costruzione a causa diversi tipi di cellule possono essere incorporati in o rivestiti sui moduli prima di essere imballate insieme per formare tessuti complessi. Ci sono tre passi principali per rendere i moduli: (1) neutralizzare il collagene e l&#39;incorporamento di cellule in esso, (2) gelificazione il collagene nel tubo e tagliare i moduli e (3) rivestimento dei moduli con le cellule endoteliali.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Creazione di micro-tessuti con gel di collagene cilindriche, chiamati moduli, che contengono cellule embedded e la cui superficie è rivestita con le cellule endoteliali.

Fabbricazione di micro-tessuti utilizzando i moduli di Collagene Gel cellule contenenti

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.6K Views.  University of Toronto. Creazione di micro-tessuti con gel di collagene cilindriche, chiamati moduli, che contengono cellule embedded e la cui superficie è rivestita con le cellule endoteliali.

Video: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Ricerca

Bioingegneria

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturate collagene fili<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via Photo-triggered ibridazione di Caged collagene Mimetic peptidi

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Umana Cartilagine Tessuto Fabrication Uso tridimensionale Tecnologia Inkjet Printing

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tissue tritata in Compressed Collagene: A Biotransplant cellulare contenente per la riparazione singolo-messa in scena Ricostruttiva

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparazione e fotoacustico Analisi dei veicoli cellulari contenenti oro Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formare giganti di dimensioni Polymersomes Uso reidratazione Gel-assistita

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabbricazione e caratterizzazione dei fantasmi di tessuto ottico contenente macrostruttura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparazione, purificazione e l'uso dei liposomi contenenti acidi grassi

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Assorbimento di nuove nanoparticelle rivestite con lipidi contenenti Falcarindiol di cellule staminali mesenchimali umane

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Produzione microfluidica di liposomi sensibili alla temperatura contengono lysolipid

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...