Gli autori riconoscono notevolmente la piattaforma PICT IBiSA presso l&#39;Institut Curie (CNRS UMR144). Questo lavoro è stato finanziato da sovvenzioni dal: Consiglio europeo della ricerca per AM.LD (Strapacemi 243.103), l&#39;Associazione Nationale pour la Recherche (ANR-09-piri-0027-PCVI), la fondazione InnaBiosanté (Micemico) a MP e AM. LD e il giovane ricercatore sovvenzione CER Strapacemi a AM.LD.

Nota importante: Questo protocollo presuppone che lo stampo contenente la forma per i microcanali desiderato è stato già fatto. Ulteriori informazioni sulla preparazione dello stampo è stato già pubblicato 10. Questo protocollo presuppone inoltre che la cultura DC midollo osseo è noto. 1. Chip Fabrication <ol><li> Mescolare olio PDMS e l&#39;agente indurente in un rapporto in peso 10:1 in una tazza di plastica. Mescolare accuratamente entrambi i composti. </li><li> Gettate la miscela sopra microcanali cuscinetto stampo. L&#39;altezza totale deve essere compresa tra 0,5-1 cm. </li><li> Rimuovere le bolle d&#39;aria in una campana barattolo vuoto durante 1 ora. </li><li> PDMS indurisce in stampo ponendo quest&#39;ultimo in stufa per 2 ore a 65 ° C. </li><li> Una volta che il PDMS è a temperatura ambiente, tagliare un grosso pezzo attorno alla struttura con una lama chirurgica e sfilarlo dallo stampo (Figura 1A). </li><li> Praticare fori dove verranno iniettate cellule (in genere 2 millimetri) e ridimensionare il PDMScon una lama chirurgica per adattarsi alle dimensioni di piatti con fondo di vetro, in cui sarà valutata la migrazione (Figura 1B). Prima di procedere, rimuovere la polvere residua dal piatto utilizzando salviette per la pulizia. Ciò favorisce il legame di PDMS al vetro descritto nella fase 1.10. </li><li> Pulire il chip PDMS ridimensionata contenente i canali attaccando e peeling nastro adesivo sui lati del dispositivo. </li><li> Sonicare i pezzi PDMS 30 sec a 70% di etanolo per rimuovere polveri e PDMS piccole particelle. Asciugare rapidamente poi soffiando aria pulita. </li><li> Attivare i PDMS (strutture verso l&#39;alto) e piatti della cultura in aereo (o di ossigeno) durante il trattamento al plasma 30 sec a 300 mTorr. </li><li> Posizionare entrambe le superfici attivate in contatto per attaccare definitivamente i PDMS al substrato. Se necessario, utilizzare pinze metalliche per premere leggermente sopra le PDMS per forzare il contatto tra il polimero e il vetro del piatto (Figura 1C). </li><li> Incubare il chip in forno a 65 ° C for 1 hr per rafforzare l&#39;associazione. </li></ol> 2. Rivestimento di microcanali <ol><li> Attivare l&#39;intera struttura da plasma di aria a 300 mTorr per 1 min. Ciò promuove l&#39;ingresso di liquido nei canali al passaggio successivo. </li><li> Riempire rapidamente i fori di ingresso del chip con fibronectina a 10 mcg / ml in acqua. Altri substrati quali PEG possono essere utilizzati per modificare l&#39;aderenza delle cellule alle pareti del canale. Prestare attenzione che il liquido si diffonde in tutta la struttura. Questo può essere facilmente controllato da occhi sotto la luce normale o utilizzando un microscopio in campo chiaro regolare. NOTA: In molto piccole strutture, in cui la diffusione è più difficile, entrata di liquido nei canali può essere forzato inserendo la struttura in una campana vaso vuoto per almeno 15 min. Per verificare l&#39;efficienza del rivestimento substrati fluorescenti possono essere utilizzati (Figura 2). </li><li> Incubare 1 ora a temperatura ambiente per permettere adsorbimento della fibronectina o qualsiasi altro rivestimento substrate alle pareti dei canali. </li><li> Lavare le strutture 3x con PBS per rimuovere il substrato nonbound. </li><li> Dopo il lavaggio, procedere al protocollo 3 o conservare il chip a 4 ° C per un uso successivo (24 ore al massimo). </li></ol> 3. Cella di carico <ol><li> Rimuovere la PBS dalla piastra e riempire il microsistema con mezzo cella. Lasciare incubare per 1 ora a saturare i canali con il mezzo. NOTA: Per gli esperimenti che coinvolgono farmaci come gli inibitori della molecola, si consiglia di Preincubare i canali con un mezzo contenente il farmaco alla giusta concentrazione. Inoltre, alcuni farmaci tendono a solubilizzare nella struttura PDMS che riduce la concentrazione efficace. Alcune soluzioni sono state proposte per contrastare questo problema 11-12. </li><li> Togliere DC galleggianti e recuperare cellule semi-aderenti mediante flussaggio con terreno di coltura. Contare le cellule utilizzando un emocitometro. NOTA: Per l&#39;imaging nucleo, un Hoechst 33342 colorazione può essere raggiunto in precedenza dalla incubating 2 x 10 6 cellule durante 30 min con il colorante a 200 ng / ml in terreno completo. Lavare due volte mediante centrifugazione per rimuovere l&#39;eccesso di colorante prima di proseguire il protocollo. </li><li> Centrifugare le cellule a 300 xg per 5 min (tempo e velocità possono essere diverse a seconda del tipo di cellula) e scartare il mezzo. Diluire il pellet per raggiungere una concentrazione di 20 x 10 6 cellule / ml. </li><li> Rimuovere l&#39;eccesso di mezzo dalla piastra e vuotare i fori di ingresso nella struttura PDMS utilizzando una micropipetta. Riempire le voci con 5 ml di soluzione di cellule per raggiungere un ammontare di 1 x 10 5 cellule in ogni foro di entrata. NOTA: elevata densità cellulare è necessario per favorire il contatto delle cellule con i canali. Bassa densità cellulare può risultare basso numero di cellule all&#39;interno canali e fallimento dell&#39;esperimento. </li><li> Incubare il microchip per 30 min a 37 ° C in incubatore. Aggiungere 2 ml di terreno completo al piatto esperimento. NOTA: L&#39;intera struttura PDMS deve be coperti per evitare l&#39;essiccazione delle cellule durante l&#39;esperimento. Se 2 ml non è sufficiente, aggiungere altro mezzo per coprire completamente la struttura PDMS. </li></ol> 4. Imaging <ol><li> Pulire la superficie inferiore esterna dei piatti con le salviettine prima di posizionare le piastre al microscopio. </li><li> Per analizzare la migrazione in gran numero di celle, utilizzare ingrandimento 10X e largo illuminazione in un campo di CO 2 e temperatura dotate di video-microscopio (Figura 3). Per facilitare il monitoraggio delle cellule, Hoechst colorazione e ai raggi UV può essere utilizzato (vedi punto 3.2). La migrazione può essere analizzata anche con microscopia confocale al fine di ottenere una maggiore risoluzione di strutture cellulari durante la migrazione. </li><li> Per microscopia time-lapse a 10X, scegliere una frequenza di tempo in funzione della velocità di cella attesi (tipicamente 2 min per cellule dendritiche migrano con una velocità di 5 micron / min). Nota: Il protocollo qui descritto fa not comprendono l&#39;analisi dei parametri di migrazione cellulare. Alcuni punti sono elencati nella discussione per consigliare i lettori su come procedere per estrarre informazioni da filmati time-lapse. </li></ol>

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>L&#228;mmermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Schor, S. L., Allen, T. D., Harrison, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+through+three-dimensional+gels+of+native+collagen+fibres:+collagenolytic+activity+is+not+required+for+the+migration+of+two+permanent+cell+lines.">Cell migration through three-dimensional gels of native collagen fibres: collagenolytic activity is not required for the migration of two permanent cell lines.</a> J. Cell. Sci. 41, 159-175 (1980).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decisions+about+dendritic+cells:+past,+present,+and+future.">Decisions about dendritic cells: past, present, and future.</a> Annu. Rev. Immunol. 30, 1-22 (2012).</li><li>Faure-Andr&#233;, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+by+CD74,+the+MHC+class+II-associated+invariant+chain.">Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.</a> Science. 322 (5908), 1705-1710 (2008).</li><li>Renkawitz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+force+transmission+in+amoeboid+cell+migration.">Adaptive force transmission in amoeboid cell migration.</a> Nat. Cell Biol. 11 (12), 1438-1443 (2008).</li><li>Jacobelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confinement-optimized+three-dimensional+T+cell+amoeboid+motility+is+modulated+via+myosin+IIA-regulated+adhesions.">Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.</a> Nat. Immunol. 11 (10), 953-961 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchinson, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 7 (12), 1783-1790 (2007).</li><li>Moreau, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+situ+cytometry+uncovers+T+cell+receptor+signaling+during+immunological+synapses+and+kinapses+in+vivo.">Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.</a> Immunity. 37 (2), 351-363 (2012).</li><li>Heuz&#233;, M. L., Collin, O., Terriac, E., Lennon-Dum&#233;nil, A. M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+in+confinement:+a+micro-channel-based+assay.">Cell migration in confinement: a micro-channel-based assay.</a> Methods Mol. Biol. 769, 415-434 (2011).</li><li>Ren, K., Zhao, Y., Su, J., Ryan, D., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+for+modifying+poly(dimethylsiloxane)+to+be+airtight+and+resistive+against+absorption+of+small+molecules.">Convenient method for modifying poly(dimethylsiloxane) to be airtight and resistive against absorption of small molecules.</a> Anal. Chem. 82 (14), 5965-5971 (2010).</li><li>Ren, K., Dai, W., Zhou, J., Su, J., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Teflon+microfluidic+chips.">Whole-Teflon microfluidic chips.</a> PNAS. 108 (20), 8162-8166 (2011).</li><li>Maiuri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+World+Cell+Race.">The first World Cell Race.</a> Curr Biol. 22 (17), 673-675 (2012).</li></ol>

Gli autori non hanno nulla da rivelare.

Qui si descrive un dispositivo composto di microcanali come metodo per studiare le proprietà migratorie di gran numero di cellule in singoli esperimenti. Questo sistema sperimentale imita i vincoli ambientali confinati trovati nei tessuti da cellule migranti endogene. Tuttavia, forzando la migrazione in una sola dimensione, facilita inseguimento cella automatica e l&#39;estrazione di measurables (Figura 5). Mostriamo anche che il nostro dispositivo è compatibile con microscopia a fluorescenza e può p...

La migrazione è una funzione cellulare complessa che è importante per molti processi fisiologici negli organismi multicellulari, incluso lo sviluppo, le risposte immunitarie e la rigenerazione dei tessuti. Inoltre, alcune situazioni patologiche come invasione tumorale e metastasi basano sulla motilità cellulare 1. Per queste ragioni, la migrazione cellulare è diventato un importante campo di studio nell&#39;ambito della ricerca sia fondamentale e traslazionale. In vivo, la maggior parte dei tessuti sono caratterizzati da una ricca matrice extracellulare ed elevata densità cellulare. La migrazione cellulare quindi, in condizioni fisiologiche, avviene in un ambiente confinato complesso. Classicamente, molto probabilmente a causa di ragioni storiche e limiti tecnici, la migrazione delle cellule è stato studiato in sistemi 2D piatte che non riproducono molte delle proprietà ambientali presenti nei tessuti, come ad esempio confinamento. Inoltre, fattori come l&#39;adesione cellulare, che sono essenziali per la motilità in 2D, sono stati recentemente dimostrato di non essere necessapalmente necessari per la migrazione in vivo o all&#39;interno gel, suggerendo che i meccanismi che regolano la locomozione cella in 2D e in altri ambienti sono distinti 2. Diversi sistemi sono stati sviluppati per imitare le proprietà complesse dei tessuti, i più noti gel dell&#39;essere collagene, che mirano a ricapitolare le proprietà della composizione della matrice extracellulare 3. Qui proponiamo microcanali come metodo semplice complementare che permette la migrazione delle cellule studio in una dimensione in un ambiente confinato. In questo sistema le cellule migrano lungo microcanali in cui essi entrano spontaneamente. Cellule migranti poi acquisire la forma dei canali, adottando una geometria tubolare che molto probabilmente rafforza il loro polarità. Il movimento lineare delle cellule nei canali permette inseguimento cella automatica e l&#39;estrazione di parametri quantitativi da esperimenti. Dal punto di vista tecnico, questo sistema è facile e flessibile. Il coating delle pareti dei canali può essere manipolato, la dimensione e la forma dei canali possono essere adattati, e un gran numero di cellule può essere analizzato in singoli esperimenti. Questo sistema può essere anche in scala ingrandita effettuare medio analisi schermata gamma di molecole coinvolte nella motilità cellulare. Il protocollo qui descritto è stato standardizzato usando cellule dendritiche (DC) come modello cellulare. Queste cellule sono fondamentali per il sistema immunitario come partecipare al procedimento e la manutenzione di risposte immunitarie specifiche 4. In vitro, cellule dendritiche hanno dimostrato di migrare spontaneamente in ambienti confinati e sono quindi un buon modello per studiare la motilità cellulare in microcanali 5,6 . È importante sottolineare che questo sistema può essere esteso per analizzare la migrazione di qualsiasi altro tipo di cellula mobili come T linfociti, neutrofili, o cellule tumorali 7-9.

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">PolyDimethylSiloxane (PDMS)</td>
			<td style="width:131px;">GE Silicones</td>
			<td style="width:139px;">RTV615</td>
			<td style="width:405px;">Package of 90% silicone base and 10% curing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core sample cutter</td>
			<td>Ted Pella Int.</td>
			<td>Harris Uni-Core&#160;</td>
			<td>Diameter 2,5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass-bottom dish</td>
			<td>WPI</td>
			<td>Fluorodish FD 35-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cleaner</td>
			<td>Branson Ultrasonics</td>
			<td>Branson 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC 32 G</td>
			<td>For small samples (35 dishes). A bigger version is also available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PolyLysine grafted PEG (Pll-g-PEG)</td>
			<td>Susos</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632</td>
			<td>TOCRIS</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of cell migration in microfabricated channels

Il metodo qui descritto consente di studiare la migrazione delle cellule sotto confinamento in una dimensione. Si basa sull&#39;uso di canali microfabbricati, che impongono un fenotipo polarizzato a cellule da limitazioni fisiche. Una volta che i canali interni, le cellule hanno solo due possibilità: andare avanti o indietro. Questa migrazione semplificata in cui direzionalità è limitato facilita il tracciamento automatico delle celle e l&#39;estrazione di parametri quantitativi per descrivere il movimento delle cellule. Questi parametri includono la velocità delle cellule, cambi di direzione, e pause durante il movimento. Microcanali sono anche compatibili con l&#39;uso di marcatori fluorescenti e sono quindi adatti per studiare la localizzazione di organelli e strutture intracellulari durante la migrazione cellulare ad alta risoluzione. Infine, la superficie dei canali può essere funzionalizzato con substrati differenti, permettendo il controllo delle proprietà adesive dei canali o lo studio di haptotaxis. In sintesi, il sistema here descritto è destinato ad analizzare la migrazione di grandi numeri di cellule in condizioni in cui sia la geometria e la natura biochimica dell&#39;ambiente sono controllate, facilitando la normalizzazione e riproducibilità di esperimenti indipendenti.

In ogni esperimento la superficie del PDMS è rivestita con una molecola adattato all&#39;interesse dello studio. Figura 2 mostra canali rivestiti con una molecola fluorescente, PLL-g-PEG, prima e dopo il lavaggio (passo 2.4). Un simile esperimento permette di controllare l&#39;omogeneità del rivestimento nei canali. Dopo il caricamento delle cellule, microscopia video può essere eseguito per seguire la migrazione cellulare. Figura 3A mostra un esempio di ...

Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

Study of Cell Migration in Microfabricated Channels

Un metodo quantitativo per studiare la migrazione spontanea di cellule in un microambiente confinato unidimensionale è descritto. Questo metodo sfrutta canali microfabbricati e può essere usato per studiare la migrazione di un gran numero di cellule in condizioni diverse in singoli esperimenti.

Studio della Migrazione cellulare in canali microfabbricati

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

study-of-cell-migration-in-microfabricated-channels

Research

JoVE Journal

Bioengineering

11.8K Views.  Institut Curie.  Un metodo quantitativo per studiare la migrazione spontanea di cellule in un microambiente confinato unidimensionale è descritto. Questo metodo sfrutta canali microfabbricati e può essere usato per studiare la migrazione di un gran numero di cellule in condizioni diverse in singoli esperimenti. 

Video: Study of Cell Migration in Microfabricated Channels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Piattaforme microfabbricazione per coltura cellulare meccanicamente dinamica

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Ricerca

Bioingegneria

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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

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

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

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

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

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creazione di sfumature adesivi e solubile per la migrazione di imaging cellulare con microscopia a fluorescenza

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

Contemporaneamente Catturare immagini in tempo reale in due canali di emissione Utilizzando un Splitting sistema Dual Camera Emissione: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Utilizzando Chips microfluidica per l&#39;imaging dal vivo e studio delle risposte lesioni in<em&gt; Drosophila</em&gt; Larve

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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 ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Modelli Distinctive capillare azione da micro-canali in osso, come può migliorare reclutamento di cellule per il Restauro di grande Bony difetto

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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 ...

Methods Article

Studio della Migrazione cellulare in canali microfabbricati