The authors would like to acknowledge Drs. Jonathan Reichner and Angle Byrd for cell experiment insight. The National Science Foundation Graduate Research Fellowship Program (GRFP) supported this work.

1. 3D Mold Design e parti di ricambio <ol><li> Muffa <ol><li> Prima di lavorare, di ottenere un kit di elastomero siliconico, una camera di imaging cellulare dal vivo, un vetrino di vetro di 22 mm e un cubo di metallo alluminio lavorato con dimensioni 10,07 millimetri x 3,95 millimetri x 5,99 millimetri. Preparare la camera di cellule vive per stampaggio ponendo il vetrino nel supporto inferiore e montaggio del resto della camera come indicato dal produttore. </li><li> Successivamente, utilizzando una pinza, posizionare il cubo di metallo alluminio lavorato al centro della custodia camera di imaging cellulare dal vivo e sulla parte superiore del vetrino, e poi mettere da parte. </li><li> Mescolare soluzioni elastomero siliconico secondo il protocollo del produttore per fare 5 ml di elastomero. </li><li> Utilizzando un laboratorio spatola monouso, versare la soluzione elastomero siliconico in configurazione camera di imaging cellulare dal vivo e garantire di non muoversi messo lavorato cubo di metallo alluminio. Posizionare il sistema sul banco di laboratorio, in una posizione sicura O / N per curare. </li><li> La mattina dopo, decostruirela camera di imaging cellulare dal vivo, come raccomandato dal costruttore e tirare fuori la muffa con pinze. Utilizzando pinze, estrarre con attenzione il cubo di metallo alluminio lavorato da muffe. Vai al lavello e lavare lo stampo con acqua deionizzata. Mettere lo stampo su un tovagliolo di carta per asciugarsi. </li><li> Una volta asciutto, di un coltello multiuso mio hobby, tagliare fessure attraverso lo stampo, distanziati 2,34 millimetri di distanza l&#39;uno estremità longitudinale, all&#39;interno dello stampo in silicone. Assicurarsi che i soggiorni di stampo in un luogo sicuro e asciutto fino a quando si è pronti a costruire il sistema per un esperimento. </li></ol></li><li> Idrofili e idrofobi vetro coprioggetto Preparazione <ol><li> Per consentire la matrice di collagene di aderire ad una superficie, creare coprioggetto idrofili: <ol><li> Usando una pipetta monouso, misurare 150 ml di 3-amminopropil-trimetossisilano e versare la soluzione in un tubo da 50 ml. Aggiungere 30 ml di etanolo al 100% al tubo 50 ml con una seconda pipetta monouso e chiudere il coperchio. Vortex la soluzione per 2 minuti, assicurando la completa miscelazione. Pla nostra soluzione in una capsula di Petri di vetro e mettere da parte. </li><li> Versare 15 ml di alcol etilico al 100% in un secondo piatto di Petri e mettere da parte (assicuratevi di etichettare i piatti). </li><li> Utilizzando una nuova pipetta monouso, misurare 30 ml di acqua deionizzata e versare la soluzione in una provetta da 50 ml. Successivamente, utilizzando una nuova misura pipetta usa e getta su 1.875 ml di glutaraldeide e versare la soluzione nello stesso tubo da 50 ml e chiudere il coperchio. Vortex la soluzione per 2 minuti, assicurando la completa miscelazione. Versare il composto in terza piastra di Petri, e mettere da parte. </li><li> Utilizzando pinze, togliere un 22 millimetri coprioggetto di vetro rotondo e risciacquare entrambi i lati con 100% di miscela di etanolo con una pipetta monouso. </li><li> Inserire coprioggetto di vetro risciacquati in 3-amminopropil-trimetossisilano con 30 ml di etanolo al 100% piatto soluzione e lasciar riposare in soluzione per 5 min. </li><li> Con una pinza, estrarre coprioggetto di vetro e sciacquare di nuovo con soluzione di etanolo al 100% con la pipetta monouso. </li><li> Goccia coprioggetto di vetro risciacquato in 1.875ml di glutaraldeide e 30 ml di miscela di acqua deionizzata e mettere da parte per 30 min. </li><li> Dopo 30 minuti, rimuovere coprioggetto di vetro con una pinza e sciacquare con acqua deionizzata e collocarlo su asciutto O tessuto / N ad asciugare a temperatura ambiente. </li><li> Ripetere immersione coprioggetto e in movimento per il numero di lamelle, se necessario con le stesse soluzioni generate. </li></ol></li><li> Fai coprioggetto idrofobiche dal protocollo data. <ol><li> Aggiungere 500 pl di tridecafluoro-1,1,2,2-tetrahydrooctyl, 100 ml di acido acetico e 19,4 ml di esano in una provetta da 50 ml e agitare per 2 min. Versare la soluzione in vetro piastra di Petri e mettere da parte. </li><li> Utilizzando pinze pulite, togliere un vetrino di vetro e cadere in soluzione mista preparata per 2 minuti. Dopo 2 minuti sono passati, togliere vetrino di vetro con una pinza e sciacquare con acqua deionizzata con una usa e getta pipetta e luogo coprioggetto su asciutto O tessuto / N ad asciugare a temperatura ambiente. Conservare vetrino in piatti di plastica Petri fino all&#39;uso. </li><li> Ripetere coprioggetto DIPPzione e spostando a molti coprioggetto come necessario con le stesse soluzioni generati. </li></ol></li></ol></li></ol> Assemblea 2. Mold <ol><li> Prima di utilizzare stampi, usando una pipetta monouso lavare lo stampo con il 90% di alcool etilico su un contenitore per rifiuti chimici. Quindi, posizionare lo stampo in una capsula di Petri riempita con acqua deionizzata e lasciar riposare O / N. </li><li> Prendete muffa dalla soluzione con pinze e posto su un telo ad asciugare prima di avviare il gruppo di forma. </li><li> Mentre lo stampo è aria di essiccazione, tagliare i coprioggetti idrofili quadrata in due rettangoli leggermente più grandi 3,95 millimetri x 5,99 millimetri utilizzando uno strumento diamante scribing alta precisione. Far scorrere ogni vetrino taglio rettangolo nelle fessure tagliate nello stampo in silicone. </li><li> Vibrazione dello stampo capovolto e applicare grasso per vuoto lungo il fondo dello stampo in silicone con una pipetta monouso. Avanti, capovolgere lo stampo posteriore lato destro in alto e premere il fondo dello stampo sulle coperture in vetro idrofili circolarilabbro per creare una tenuta. </li><li> Con guanti monouso, prendere lo stampo montato e mettere in un piatto e getta Petri e poi in un bio-cappuccio. Accendere lampada UV Bio-cappuccio per 1 ora per sterilizzare lo stampo prima che si verifichi la sperimentazione. </li></ol> 3. Il collagene miscela e 3D Matrix <ol><li> Prima di preparare la miscela di collagene, macchia e preparare le cellule per l&#39;imaging secondo protocolli standard 28. </li><li> Utilizzando tecniche di laboratorio standard e protocolli 29,30, in un bio-hood, mescolare carta cellulari e chemiotattico in un tubo da 15 ml alla concentrazione chemoattractant desiderata. Pipettare 5 ml di soluzione specifica cella media-chemiotattico concentrazione in una provetta da 15 ml e spostare la soluzione in un bagno d&#39;acqua riscaldata. </li><li> Usando una pipetta, estrarre 5 ml di supporti cellulari solo in un secondo tubo da 15 ml e posto in un bagno d&#39;acqua riscaldata per riscaldare la soluzione per esperimenti di microscopia. </li><li> In un bio-hood, pipetta 30 ml di 10x tampone fosfatosoluzione ed (PBS) in micro provetta da centrifuga. Aggiungere 6 ml di 1 N di idrossido di sodio (NaOH) in stessa provetta da centrifuga micro e vortex per 15 sec. </li><li> Ottenere soluzione coda di ratto collagene I dal frigo e passare al bio-cappa utilizzando tecniche di laboratorio standard e protocolli 29. Assicurarsi che il collagene è ancora freddo. Pipetta 168,3 ml di collagene nella stessa provetta da centrifuga micro. </li><li> Successivamente, aggiungere 18 ml di microsfere carbossilato modificato fluorescenti giallo-verde con una pipetta alla stessa provetta da centrifuga micro. Vortex provetta micro con tutte le soluzioni per 30 sec. </li><li> Aggiungere 77,7 ml di desideri miscela di cellule media-cell (circa 2 x 10 6 cellule / ml concentrazione) in micro tubo di centrifuga e miscelare con puntale per 20 sec. Se necessario, modificare la densità matrice seguendo la miscela collagene personalizzato adattato per l&#39;aggiunta di perline fluorescenti e tavolo vitalità cellulare (Tabella 1). </li><li> Dispensare 300 &amp; #181; l di miscela di collagene-cellule al centro (pozzetto 2 #) del complesso di stampo preparato. Collocare lo stampo su un piatto Petri monouso e spostarlo in un incubatore standard per 20 min a 37 ° C con 5% di CO 2. </li><li> Togliere sistema da incubatore e mettere di nuovo in bio-cappuccio. Utilizzando pinze, rimuovere entrambi i vetrini idrofobici fissando sulla parte superiore di ogni vetrino e tirando alto e lontano dallo stampo. </li><li> Spostare l&#39;intero sistema alla microscopia e immagine desiderata per assicurare lati dello stampo collagene sono ancora diritto per consentire un gradiente planare diffusione. </li><li> Con il sistema ancora sotto il microscopio, utilizzando un 100 microlitri monocanale pipetta, aggiungere 100 ml di supporti cellule nel pozzetto 1 #. Successivamente, utilizzando una 100 pl monocanale pipetta aggiungere 100 ml di media cella con concentrazione chemoattractant desiderato nel bene # 3. </li><li> Subito dopo l&#39;aggiunta di soluzioni in entrambi i pozzi, iniziare imaging. </li></ol> 4. Imaging e DiffusioneModellismo <ol><li> Se si vuole trovare il coefficiente di diffusione per la densità del collagene specifico per calcolare la concentrazione specifica, seguire il protocollo di seguito: <ol><li> Seguire il protocollo di cui sopra dal titolo &quot;miscela di collagene e 3D Matrix&quot;, invece di generare supporti cella con concentrazione chemoattractant desiderata, fare 5 micron rhodamine in un tubo da 15 ml con calcoli e protocolli 30 standard. </li><li> Immagine matrici collagene 3D con un sistema confocale montato su un microscopio invertito utilizzando un laser ad argon (488 nm) per catturare la fluorescenza. Immagine ogni 2 o 3 secondi per un massimo di 7 ore. </li></ol></li><li> Per Modeling Diffusione: Usando il modello matematico mostrato di seguito, scrivere un codice software di calcolo per l&#39;elaborazione e l&#39;analisi di questi passaggi generali. <img alt="Equazione 1" src="/files/ftp_upload/52948/52948eq1.jpg" /><ol><li> Importazione di immagini in software di calcolo per ridimensionamento e normalizzazione da parte della massima intensità. Converti immagini in un colore grigio-mappa dell&#39;immagine per selezionare il bordo. Scegliere un asse lungo il centro dell&#39;immagine, a destra del centro e alla sinistra del centro. </li><li> Usando il codice del software di calcolo, tracciare l&#39;intensità normalizzata, insieme con la vera intensità e la differenza tra i due. </li><li> Quindi, immettere i parametri necessari per il montaggio (concentrazione, tempo e lunghezza). </li><li> Tracciare la concentrazione normalizzata in funzione del tempo e il profilo di concentrazione attraverso l&#39;asse x con un&#39;approssimazione polinomiale ai dati. </li><li> Calcolare il coefficiente di diffusione utilizzando il codice software di calcolo. </li></ol></li></ol> 5. Misure sperimentali <ol><li> Riferendosi di nuovo alla equazione di diffusione, riscrivere l&#39;equazione per determinare la concentrazione specifica in qualsiasi posizione all&#39;interno del sistema, come mostrato in seguito. <img alt="Equazione 2" src="/files/ftp_upload/52948/52948eq2.jpg" /> Dove: L = Lunghezza del the matrice di collagene x = posizione sotto inchiesta D = coefficiente di diffusione t = tempo trascorso </li><li> Durante gli esperimenti di chemiotassi, assicurarsi di registrare il tempo specifico che il momento chemochina è stato aggiunto al sistema. Una volta trovato il movimento migrazione delle cellule, assicurarsi di registrare confocale time-lapse, e prendere nota del tempo specifico e la posizione dal bordo dove si è verificato l&#39;imaging. </li><li> Durante l&#39;analisi dei dati, inserire il noto tempo trascorso, la posizione e il coefficiente di diffusione per la cella specifica nell&#39;equazione per trovare la concentrazione specifica normalizzata in qualsiasi punto all&#39;interno del collagene sia per diffusione e esperimenti sulle cellule. </li></ol> 6. Monitoraggio migrazione cellulare Utilizzando TFM <ol><li> Per monitorare la migrazione delle cellule utilizzando tecniche TFM / DVC seguire il protocollo di seguito: <ol><li> Dopo l&#39;aggiunta di concentrazione chemoattractant desiderato nel pozzetto 3 #, avviare l&#39;imaging delle matrici di collagene 3D con un sistema confocale montato su un micr invertitaoscope per trovare una cella per indagare. </li><li> Una volta che una cellula in movimento viene trovato, posizionare la cella al centro del campo visivo e utilizzare un motore piezoelettrico (all&#39;immagine in asse z), un&#39;immagine ogni 1 o 2 min. </li><li> Per calcolare la generazione di forza cellulare e deformazione, generare un codice di calcolo per trovare gli spostamenti delle perline fluorescenza seguenti protocolli TFM / DVC 31. </li></ol></li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory 2e. , (2010).</li><li>Bar-Kochba, E., Toyjanova, J., Andrews, E., Kim, K., Franck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fast+Iterative+Digital+Volume+Correlation+Algorithm+for+Large+Deformations.">A Fast Iterative Digital Volume Correlation Algorithm for Large Deformations.</a> Exp. Mech. In press, (2014).</li><li>Shenoy, V., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+Macromolecules+in+Collagen+and+Hyaluronic+Acid,+Rigid-Rod-Flexible+Polymer+Composite+Matrixes.">Diffusion of Macromolecules in Collagen and Hyaluronic Acid, Rigid-Rod-Flexible Polymer Composite Matrixes.</a> Macromolecules. 28 (26), 8751-8758 (1995).</li><li>Leddy, H., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Molecular+Diffusion+in+Articular+Cartilage+Measured+using+Fluorescence+Recovery+after+Photobleaching.">Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching.</a> Ann. Biomed. Eng. 31 (7), 753-760 (2003).</li></ol>

The authors declare that they have no competing financial interests.

Le fasi più critiche per esperimenti di diffusione di successo con o senza cellule sono: impostare correttamente il gruppo di forma; sviluppare la manualità necessaria per evitare danni durante l&#39;estrazione dei vetrini idrofobici; garantendo di trovare una buona linea di partenza lineare per calcolare correttamente il coefficiente di diffusione; correggere i calcoli sperimentali di collagene e chemiotattico; utilizzare correttamente del sistema di imaging cellulare dal vivo per assicurare matrice non si secca; e m...

Il movimento preferito di cellule verso un gradiente di concentrazione, noto come chemiotassi, svolge un ruolo importante in processi patologici e fisiologici del corpo. Tali esempi sono la pelle e le mucose guarigione delle ferite 1, morfogenesi 2, infiammazione 3, e la crescita tumorale 4,5. E &#39;stato anche dimostrato che le cellule tumorali possono migrare attraverso due strategie cellule migratorie individuali e collettive 6. Inoltre, i meccanismi di instabilità diffusionali possono indurre la separazione delle cellule singole o cluster da un corpo / oggetto tumorale e quindi in grado di emigrare verso una fonte di sostanze nutritive e di invadere le zone più ampie e tessuti 7 così. Inoltre, è stato dimostrato che i meccanismi di migrazione diverse possono essere attivi in 2D e in 3D, a causa di diversi ruoli delle molecole di adesione 8. Pertanto, una mossa per fisiologicamente rilevanti in vitro per indagare motilità cellulare in un measureable e modo semplice è di importanza nella comprensione movimento delle cellule fenomeni 9. Purtroppo, la difficoltà nell&#39;analisi migrazione cellulare, un completo test chemiotassi quantificabile solito richiede un metodo lungo laboriosa, fondata sulla misurazione della motilità cellulare e fenomeni di trasporto modelli imparziale. Approcci sperimentali passato per indagare chemiotassi cellulari includono la camera di Boyden 10 e il sotto dosaggio agarosio 11. Tuttavia, all&#39;interno di questi primi test, esperimenti di migrazione delle cellule non hanno controllano il movimento in relazione al tempo. Di più, importante, i gradienti di concentrazione usati per gli esperimenti sono stati non ben definiti o completamente compresi mentre solo sostenere la segnalazione per non più di qualche ora. Inoltre, presto chemiotassi tentativi camera limitato la migrazione delle cellule a due dimensioni e non permettono di monitorare la cinetica della migrazione 12. Guardando la camera di Boyden, un saggio finalenon consentirebbe al ricercatore di osservare la migrazione visivamente e non ha potuto differenziare direttamente chemiotassi (movimento direzionale) da chemochinesi (movimento casuale). Inoltre, diverse variabili differenze nella dimensione dei pori e lo spessore delle membrane-reso la camera molto difficile da riprodurre facilmente e nascosti reazione migrante delle cellule di chemochine 13,14. Con la nuova comprensione di microfluidica, nuove camere e micro-dispositivi sono stati studiati come strumento per indagare locomozione cella in condizioni di flusso interstiziali o chemiotassi 15,16. In questi nuovi dispositivi, sono stati introdotti e studiati, come l&#39;effetto di sollecitazione di taglio su una cella 17,18 nuove metriche cellulari. Purtroppo, le camere chemiotassi microfluidica passati e attuali limitati studi di migrazione cellulare a substrati-an 2D battuta d&#39;arresto importante dal momento che molti processi biologici, tra cui l&#39;invasione delle cellule tumorali e metastasi, e immigrazione cellulare mune, comporta migrazione 3D. Camere-dove osservazione diretta di una soluzione chemiotattico è in contatto con un gel 3D contenente cellule sono anche stati riportati anche 19,20. Queste camere hanno due scomparti, uno contenente un fattore chemiotattico e una contenente cellule, sono uniti l&#39;uno accanto all&#39;altro in orizzontale 21 o come anelli concentrici 22. Questi sistemi sono puntati nella giusta direzione, ma non mantengono un sistema di chemiotassi per un periodo di tempo prolungato. Inoltre, i ricercatori hanno esaminato diffusività attraverso membrane di collagene nelle cellule dialisi, nonché la diffusione delle molecole traccianti attraverso campioni di collagene sottoposta a pressione idrostatica 23-25. Alcuni esperimenti di diffusione in gel di collagene affidano a modificazioni fisiche e chimiche del gel utilizzando campi magnetici e incorporazione chimiche 26. Un metodo comune per la modellazione di diffusività in collatessuti azo tati si basa sulla fluorescenza del punto photobleaching continuo. Questo metodo ha rivelato anisotropia nei coefficienti di diffusione di macromolecole nei tessuti collageni orientati. Tuttavia, photobleaching è stato usato nella cartilagine articolare e non collagene matrici. Mentre simile, gli esperimenti di modellazione necessari devono essere effettuati tramite specificamente comprendere il coefficiente di diffusione del gel di collagene. Ancora più importante, i sistemi non utilizzano un metodo per misurare generazione di forza cella. Sfortunatamente, la maggior parte dei sistemi sembrano mancare uno o due elementi fondamentali per un sistema ideale: il che consente di inseguimento cellulare, una comprensione gradiente di diffusione con un fattore chemiotattico attraverso la matrice, un insieme relativamente semplice con una facilità di riproducibilità, la minimizzazione interazioni cellula-cellula, e la capacità di misurare unità dimensionali per quantificazione (cioè, velocità, forza, concentrazione specifica). Moghe et al. </em&gt; 27 ha proposto un sistema che soddisfaceva la maggior parte di questi requisiti in cui le cellule sono state inizialmente disperse nel gel anziché concentrati sulla superficie del filtro, ma era difficile misurare le forze che la cellula genera. A questo proposito, vi presentiamo un sistema planare gradiente di diffusione di indagare chemiotassi in una matrice di collagene in 3D, che permette di superare i limiti moderni camera di diffusione dei dosaggi esistenti, che si basa sulla microscopia time-lapse, associato a tecniche di analisi di immagine per la misurazione delle cellule forze in un ambiente 3D. Questo protocollo fornisce un modo semplice, ma innovativo di creazione di una semplice camera di diffusione 3D che può essere usato per studiare chemiotassi 3D in celle diverse.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Silicone elastomer kit</td>
			<td>Dow Corning Corp</td>
			<td>182 SIL ELAST KIT .5KG</td>
			<td>a two-part misture with a 10:1 mix</td>
		</tr>
		<tr>
			<td>Live cell imaging chamber</td>
			<td>Live Cell Instrument</td>
			<td>CM-B18-1</td>
			<td>CMB for 18mm round coverslips</td>
		</tr>
		<tr>
			<td>22mm Glass Coverslip</td>
			<td>Fisher Scientific</td>
			<td>NC0180281</td>
			<td>Neuvitro Corp. cover slip 22mm 1.5</td>
		</tr>
		<tr>
			<td>Machined aluminum metal cube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hobby utility knife</td>
			<td>X-Acto</td>
			<td>X3201</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(aminopropyl) trimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Polysciences, Inc</td>
			<td>00216A-10</td>
			<td>Glutaraldehyde, EM Grade, 8%</td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>Standard floor model and tabletop centrifuges</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747A</td>
			<td>Reusable Petri Dishes: Complete (60 x 15mm)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327-379</td>
			<td>Fine Point Forceps</td>
		</tr>
		<tr>
			<td>Cover glasses</td>
			<td>Fisher Scientific</td>
			<td>12-518-105A</td>
			<td>Rectangle; 30 x 22mm; Thickness No. 1</td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl</td>
			<td>Gelest</td>
			<td>SIT8174.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td>Acetic acid ACS reagent, &#8805;99.7%</td>
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		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td>Hexane</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>anhydrous, 95%</td>
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			<td>Ethyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>High-precision diamond scribing tool</td>
			<td>Lunzer</td>
			<td>PV-081-3</td>
			<td>Straight extended tip scribe, .020&#34; (.50mm) diameter by .200&#34; (5.0mm) tip length</td>
		</tr>
		<tr>
			<td>Vacuum grease</td>
			<td>Dow Corning</td>
			<td>14-635-5C</td>
			<td>High-Vacuum Grease</td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-49D</td>
			<td>15 ml conical centrifuge tubes with hydrophobic, biologically inert surface</td>
		</tr>
		<tr>
			<td>10X phosphate buffered solution</td>
			<td>Fisher Scientific</td>
			<td>BP399-500</td>
			<td>1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer</td>
		</tr>
		<tr>
			<td>1N sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>38215</td>
			<td>Sodium hydroxide concentrate</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>Rat tail&#160;</td>
		</tr>
		<tr>
			<td>Micro centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>02-681-332</td>
			<td>Volume: 2.0 ml; O.D. x L: 13 x 40 mm; sterile; single-wrapped</td>
		</tr>
		<tr>
			<td>Carboxylate-modified microspheres</td>
			<td>Invitrogen</td>
			<td>&#160;F-8813</td>
			<td>Carboxylate-modified microspheres, 0.5 &#181;m, yellow-green fluorescent (505/515), 2% solids</td>
		</tr>
		<tr>
			<td>Rhodamine</td>
			<td>Sigma-Aldrich</td>
			<td>83689</td>
			<td>Rhodamine B for fluorescence</td>
		</tr>
	</tbody>
</table>

planar gradient diffusion system to investigate chemotaxis in a 3d collagen matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

La capacità di questo test per valutare con precisione la migrazione della cella si basa su una buona configurazione del sistema. Pertanto, è fondamentale per fare in modo di progettare lo stampo sistema di diffusione in modo accurato e fare molta attenzione nel porre coprioggetto sia idrofobe e idrofile, come illustrato nella figura 1. Se il sistema è correttamente progettato e durante la fase di modellazione diffusione garantendo trovare molto buona linea di partenza lineare, si è in grado di ragg...

Watch this Scientific Journal Video about Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix at JoVE.com

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

planar-gradient-diffusion-system-to-investigate-chemotaxis-3d

Research

JoVE Journal

Bioengineering

8.4K Views.  California State University, Long Beach. Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Video: Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

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 ...

Ricerca

Bioingegneria

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

Concentric Sistema Gel per studiare il ruolo biofisica di Matrix Microenvironment sulla migrazione Cell 3D

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

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. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradiente di deformazione Chip per comportamenti stimolanti cellulari in cella-carichi idrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

Un metodo per la determinazione e la simulazione di permeabilità e la diffusione in un modello 3D del tessuto in una membrana inserire sistema per piastre multi-pozzetto

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.