Gli autori ringraziano Ronald F. Renzi e Michael S. Bartsch per i loro contributi alla progettazione e sviluppo di dispositivi di DMF e l&#39;hub DMF. Questa ricerca è stata interamente sostenuta dal Laboratorio di Regia Programma di ricerca e sviluppo presso i Sandia National Laboratories. Sandia è un laboratorio multi-programma gestito e gestito da Sandia Corporation, una consociata interamente controllata di Lockheed Martin Corporation, per il Dipartimento della National Nuclear Security Administration di Energia degli Stati Uniti sotto contratto DE-AC04-94AL85000.

Questo protocollo generale è progettata per supportare l&#39;applicazione della piattaforma per un&#39;ampia varietà di studi; aspetti specifici allo studio rappresentativo descritta nella presente relazione vengono separati tra parentesi Figura 2 illustra uno studio rappresentativo effettuato utilizzando il protocollo.. Si noti che nel protocollo, tutte le &quot;istruire&quot; i comandi sono automatizzati attraverso l&#39;uso di script pre-determinati. Si noti anche che la Fase 2 può essere effettuata in parallelo con la fase 1 (ad esempio durante le fasi 1.7 e 1.8), e che la Fase 2.1 è spesso bypassato (perché modellata / piastre rivestite dispositivo DMF possono essere lavati e riutilizzati; vedi passo 5.2) . 1. Assemblare e popolare le camere di perfusione delle cellule <ol><li> Ricoprire le superfici interne di sterilizzato (autoclave) microcapillari (silice fusa; 679 micron od, id 534 micron; lunghezza 15 cm) con fibronectina, istruendo la pompa a siringa per disegnare una soluzione sterile di fibronectina (30 pl di 50 ug / ml) in ciascunmicrocapillari, incubando (37 ° C per 2 h), istruendo la pompa a siringa per aspirare la soluzione, e lasciando che il microcapillari secco (temperatura ambiente (RT) per 6 ore). </li><li> Collegare ogni fibronectina rivestito microcapillare (camera di perfusione) per tubi in policarbonato (500 micron od, id 100 micron) e il tubo alla pompa siringa multi-porta, utilizzando raccordi CapTite (6 camere di perfusione, 8 porte pompa siringa). </li><li> Con del nastro, fissare il corpo di ogni camera di perfusione ad un blocco di calore impostato temperatura desiderata (37 ° C). </li><li> Immergere le estremità aperte delle camere di perfusione in un serbatoio (tubo microcentifuge o micropiastra bene) le celle che contengono (P388D.1 macrofagi murini) sospese in terreno di crescita fresca (RPMI-1640, 10% FBS, 500 unità / ml di penicillina, 500 mg / ml di streptomicina) alla concentrazione desiderata (10 6 cellule / ml). </li><li> Istruire la pompa a siringa per disegnare le celle (10 ml, 10 4 cellule) in ogni camera di perfusione seguita da sufficiente air per spostare il tappo di liquido (~ 4,5 cm) alla sezione fissata al blocco di calore. </li><li> Permettono alle cellule di aderire alle superfici interne rivestite di fibronectina delle camere di perfusione (37 ° C per 1 - 2 ore). Nel frattempo, trasferire le estremità aperte delle camere di perfusione dal serbatoio cellule per un nuovo serbatoio contenente terreno di coltura fresco. </li><li> Dopo il periodo di adesione, istruire la pompa a siringa per inviare i tappi di liquidi (mezzi condizionati e cellule non collegata) da perdere, ritirarsi mezzo fresco (10 ml), e seguire con aria sufficiente per posizionare i nuovi tappi di liquido (mezzo fresco) sulla aderito cellule. </li><li> Continuare a svolgere scambi medi (cioè ripetere il punto 1.7) a intervalli regolari (ogni 2 ore) fino a quando le popolazioni cellulari sono sufficientemente equilibrato ed espansa (16 - 24 ore di coltura di micro-scala generate popolazioni di ~ 1.000 cellule / camera). </li></ol> 2. Realizzare il dispositivo DMF e montare il mozzo Architettura <ol><li&gt; Come descritto in precedenza 14,29, modello la piastra inferiore del dispositivo DMF con quarantasei ossido di indio e stagno (ITO) elettrodi (conducenti di goccia erogazione) di fotolitografia e incisione, e cappotto con 5 micron di parylene-C e 50 nm di Teflon AF. Formare la piastra superiore del dispositivo DMF mediante rivestimento di un substrato di vetro ITO nanostrutturata con Teflon-AF, come sopra. </li><li> Utilizzando telai metallici di compressione, fissare le piastre DMF in un cast polimero 14,30 con rientranze che mantengono una spaziatura 400 micron tra le piastre, questa spaziatura, unitamente al volume elettrodo azionamento (2,5 mm 2), definisce il volume delle gocce (2,5 microlitri per ogni elettrodo). Notare che DMF assemblaggio può essere realizzato con mezzi più semplici 24. </li><li> Inserto teflonato trasferimento microcapillari (360 micron od, id 100 micron; 3,5 - lunghezza 4,0 cm) nello spazio tra le piastre DMF, posizionando ciascuno di estendere al bordo del suo affine elettrodo di azionamento. </li><li> Al contrario end di ogni trasferimento apporre microcapillare un raccordo CapTite. </li><li> Innestare i connettori elettrici per fornire tensione agli elettrodi ITO. Sequenze di attivazione degli elettrodi sono automatizzate con l&#39;uso di una interfaccia elettronica controllato dal computer che esegue gli script di pre-determinati. </li><li> Opzionale: Spostare il mozzo DMF montato sul palco traslazionale di un microscopio (SZ-6145TR (Olympus, Giappone)) dotato di un dispositivo ad accoppiamento di carica (CCD) fotocamera (DCR-HC96 (Sony, Giappone)) per facilitare il monitoraggio delle goccioline 31. </li></ol> 3. Collegare le camere di perfusione al Hub DMF e stimolare le popolazioni di cellule <ol><li> Rimuovere le estremità aperte delle camere di perfusione dal serbatoio mezzo (vedere il punto 1.6) e inserirli nei raccordi CapTite fissate alle estremità del trasferimento microcapillari del mozzo DMF (vedere il punto 2.4). </li><li> Istruire la pompa a siringa per inviare i tappi di liquidi (mezzi condizionati) nelle camere di perfusione da perdere. </li><li> Instruct il mozzo DMF per disegnare stimolo (E. coli bioparticelle a 100 pg / ml in terreno fresco contenente 0.1% Pluronic F127 w / v) da un serbatoio di bordo e fornire una gocciolina di dimensione appropriata (10 microlitri) per ogni trasferimento microcapillari . Le bordo serbatoi sono costituiti da scomparti di forma cilindrica (1,5 x 1,5 cm ciascuno) e collegato all&#39;hub DMF tramite un tubo. </li><li> Istruire la pompa a siringa per disegnare lo stimolo collega al trasferimento microcapillari, e seguire con aria sufficiente per posizionare i tappi sulle popolazioni cellulari nelle camere di perfusione. </li><li> Incubare le cellule con stimolo (37 ° C per 1 - 4 ore). Optional: Cambio di stimolo fresco (cioè ripetere i passaggi 3,2-3,4) ad intervalli regolari. </li></ol> 4. Terminate stimolazione e recuperare lisati cellulari per l&#39;analisi <ol><li> Opzionale: Se è richiesto un periodo di post-stimolazione, scambiarsi gli stimoli che contengono spine liquidi per mezzo fresco (cioè ripetere i passi 3,2-30,4, sostituendo mezzo fresco di stimolo), e continuare a incubare e scambiare, se necessario. </li><li> Scambio i tappi liquido nelle camere di perfusione per la soluzione di lavaggio (Prelude diretto Lysis Buffer Modulo B) (cioè ripetere i passaggi 3,2-3,4, in sostituzione del tampone di lavaggio per stimolo), e incubare come necessario (5 min). </li><li> Scambio i tappi liquidi (tampone di lavaggio) per la soluzione di lisi (Preludio diretto Lysis Buffer Modulo A con 0,1% Pluronic F127 w / v) (cioè ripetere i passaggi 3,2-3,4, sostituendo la soluzione di lisi per stimolo), e incubare come necessario (5 min) . </li><li> Istruire la pompa a siringa per inviare i tappi liquidi (lisato cellulare) al mozzo DMF. </li><li> Smontare il mozzo DMF, rimuovere la piastra superiore del dispositivo DMF (facendo attenzione a non inclinare il piatto di lato, in quanto questo può portare a delle gocce di miscelazione), e raccogliere i lisati utilizzando una Pipetman o siringa per l&#39;analisi off-piattaforma (profili di espressione genica tramite qPCR). </li></ol> 5. Clean piattaforma hardware perRiutilizzare <ol><li> Immergere il trasferimento microcapillari e raccordi CapTite in candeggina al 10% (v / v in acqua) per 10 min a RT, sciacquare bene con acqua deionizzata e asciugare con gas azoto. </li><li> Immergere le piastre dispositivo DMF nel 10% di candeggina per 10 min a RT, sciacquare bene con isopropanolo e poi con acqua deionizzata e asciugare con azoto seguita da cottura a 160 ° C per 10 min. </li><li> Tubi in policarbonato della pompa siringa, e polimero fuso del dispositivo DMF e telaio di compressione, può essere riutilizzato senza pulizia. La camera di perfusione microcapillari vengono scartati dopo ogni esperimento. </li></ol>

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E., 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=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Abbiamo sviluppato una piattaforma automatizzata semplice e versatile per la coltura cellulare micro-scala e di esperimenti di stimolazione. La piattaforma consente di lavorare con piccoli volumi di coltura e le popolazioni di cellule (1-20 ml e 100 - 2.000 cellule, per camera); dimensioni cultura potrebbe essere ulteriormente ridotto mediante l&#39;utilizzo di microcapillari di diametro inferiore. Lavorare in queste scale riduce il costo degli studi di routine e rende realizzabili gli studi che richiedono l&#3...

Lo studio delle cellule mantenute in coltura (analisi in vitro) ha fornito preziose informazioni sui principi fondamentali e dei meccanismi molecolari che regolano i complessi sistemi biologici e la salute umana. I metodi convenzionali per la cultura, la stimolazione, e la raccolta di cellule per l&#39;analisi, che utilizzano capsule di Petri e micropiastre, sono stati progettati per lo studio di grandi popolazioni di cellule (≥ 10 5) in volumi di coltura millilitro scala (≥ 0,1 ml). Tuttavia, ci sono molti casi in cui sono disponibili soltanto quantità limitate di cellule (per esempio cellule primarie), o piccole popolazioni di cellule sono desiderabili (es. ridurre cellula-cellula variabilità nella popolazione), o reagenti necessari sono difficili da ottenere o costi proibitivi (ad es purificati fattori cellula-secrete). Tali problemi possono essere affrontati con successo dal ridimensionamento dimensione culturale, che ha il vantaggio di ridurre il consumo di tutti ireagenti necessari per l&#39;analisi in vitro 1,2. Purtroppo, attrezzature e metodi convenzionali non supportano la manipolazione precisa e riproducibile di culture micro-scala ed i sistemi automatizzati di microfluidica a base attualmente disponibili 3-11 sono troppo complesse e specializzate per l&#39;uso di routine dalla maggior parte dei laboratori. In questo rapporto, descriviamo il montaggio e l&#39;uso di una piattaforma tecnologica semplice e versatile per la cultura automatizzata, la stimolazione, e il recupero di piccole popolazioni di cellule (100 - 2.000 cellule) in volumi di micro-scala (1-20 ml). L&#39;architettura della piattaforma (Figura 1) ha un design modulare: una serie di fibronectina rivestito microcapillari (&quot;camere di perfusione delle cellule&quot; modulo) serve come sito per la creazione, la manutenzione e la stimolazione delle colture micro-scala, e una microfluidica digitale (DMF ) 12,13 dispositivo equipaggiato con il &quot;trasferimento&quot; microcapillari (modulo &quot;centrale hub&quot;) 14,15 celle rottee reagenti da e per le camere di perfusione. DMF consente all&#39;utente di affrontare individualmente multiple goccioline simultaneamente e per modificare o ri-ordinare le manipolazioni (cioè riconfigurare i treni di trattamento del campione) senza modificare l&#39;hardware del dispositivo. La sua grande flessibilità è evidente nella sua recente comparsa come una tecnologia chiave in una vasta gamma di applicazioni, tra cui la coltura cellulare 16,17, saggi enzimatici 18,19, 20,21 immunologici, analisi del DNA 22,23, trasformazione delle proteine, 24,25 clinica e trattamento dei campioni. 26,27 nostro mozzo centrale sfrutta la flessibilità inerente ai dispositivi DMF, e migliora ulteriormente attraverso l&#39;aggiunta di interfacce microcapillari, che fornisce opportunità di realizzare un sottoinsieme di manipolazioni (es. coltura cellulare) in periferico specializzato moduli, piuttosto che sul dispositivo DMF stesso. Compartimentazione dei treni di elaborazione in questo modo semplifica anche la progettazione del plaarchitettura TForm (nessun bisogno di costruire un dispositivo di DMF in grado di eseguire tutte le fasi di lavorazione) e facilita la sua evoluzione come nuove funzioni sono necessarie (semplicemente integrare nuovi moduli periferici, se necessario). Trasporto delle cellule e reagenti all&#39;interno del mozzo centrale è guidato da elettrowetting forze generate mediante attivazione sequenziale di elettrodi all&#39;interno del dispositivo di DMF 13,28, trasporti verso, da e all&#39;interno delle camere di perfusione è alimentato da variazioni di pressione generate da una siringa ad alta precisione pompa. Tutti questi movimenti fluidi sono controllati tramite una semplice interfaccia elettronica e automatizzata mediante l&#39;utilizzo di script pre-determinati. Come esempio rappresentativo, dimostriamo l&#39;utilizzo della piattaforma per lo studio delle risposte trascrizionali suscitato in cellule del sistema immunitario su sfida con i batteri (Figura 2). Svolgimento di tali esperimenti sulla piattaforma ci ha permesso di lavorare con un piccolo numero di cellule (~ 1.000 per sperimentazionecondizione Tal), ridurre al minimo la variabilità esperimento a esperimento, i reagenti conservare, e ri-diretto hands-on del lavoro. Considerati i vantaggi che essa conferisce, così come la sua accessibilità e versatilità, questa piattaforma dovrebbe trovare uso in un&#39;ampia varietà di laboratori e applicazioni e rivelarsi particolarmente utile nel facilitare l&#39;analisi di cellule e stimoli che sono disponibili solo in quantità limitate.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Studio delle cellule in coltura (analisi in vitro) ha fornito indicazioni importanti in sistemi biologici complessi. I metodi convenzionali e attrezzature per analisi in vitro sono adatti per lo studio di un gran numero di cellule (≥ 10 5) in volumi millilitro scala (≥ 0,1 ml). Tuttavia, ci sono molti casi in cui è necessario o auspicabile ridimensionare dimensione coltura per ridurre il consumo delle cellule di interesse e / o reagenti richiesti per la loro cultura, la stimolazione, o la trasformazione. Purtroppo, gli approcci tradizionali non supportano la manipolazione precisa e riproducibile di culture micro-scala, e dei sistemi automatizzati microfluidica a base attualmente disponibili sono troppo complesse e specializzate per l&#39;uso di routine dalla maggior parte dei laboratori. Per affrontare questo problema, abbiamo sviluppato una piattaforma tecnologica semplice e versatile per la cultura automatizzata, la stimolazione, e il recupero di piccole popolazioni di cellule (100 - 2.000 cellule) nel volume di micro-scalas (1 - 20 l). La piattaforma è costituita da un insieme di microcapillari fibronectina-rivestito (&quot;camere di perfusione delle cellule&quot;), all&#39;interno del quale sono stabilite le culture micro-scala, mantenuti e stimolati; un dispositivo digitale di microfluidica (DMF) equipaggiati con &quot;trasferimento&quot; microcapillari (&quot;hub centrale &quot;), quali cellule rotte e reagenti da e verso le camere di perfusione; una pompa a siringa ad alta precisione, che trasportano i poteri di materiali tra le camere di perfusione e il mozzo centrale, e un&#39;interfaccia elettronico che consente di controllare il trasporto di materiali, che è coordinato e automatizzato tramite script pre-determinati. Come esempio, abbiamo utilizzato la piattaforma per facilitare lo studio di risposte trascrizionali indotte in cellule immunitarie upon sfida con i batteri. Utilizzo della piattaforma ci ha permesso di ridurre il consumo di cellule e reagenti, minimizzare la variabilità esperimento a esperimento, e ri-diretti hands-on del lavoro. Considerati i vantaggi che essa conferisce, così come la sua accessibilità e versatilità, opiattaforma ur dovrebbe trovare uso in un&#39;ampia varietà di laboratori e applicazioni, e rivelarsi particolarmente utile nel facilitare l&#39;analisi di cellule e stimoli che sono disponibili solo in quantità limitate.

A dimostrazione della piattaforma automatizzata, lo abbiamo utilizzato per realizzare uno studio in cui piccole popolazioni di cellule immunitarie sono state fatte crescere in colture di micro-scala, provocati con i batteri, e lisate per l&#39;analisi off-piattaforma di risposte pro-infiammatorie (Figura 2 ). Ognuno dei sei camere di perfusione delle cellule è stato seminato con 10 4 cellule immunitarie (P388D.1 macrofagi murini) risospeso in 10 ml di terreno di ...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Abbiamo sviluppato una coltura cellulare automatizzata e piattaforma di interrogatorio per esperimenti di stimolazione cellulare micro-scala. La piattaforma offre un controllo semplice, versatile e preciso nel coltivare e stimolare le piccole popolazioni di cellule, e recuperando lisati per le analisi molecolari. La piattaforma è adatto a studi che impiegano cellule preziosi e / o reagenti.

Una piattaforma automatizzata versatile per micro-scala Cellulari esperimenti di stimolazione

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K Views.  Sandia National Laboratories. Abbiamo sviluppato una coltura cellulare automatizzata e piattaforma di interrogatorio per esperimenti di stimolazione cellulare micro-scala. La piattaforma offre un controllo semplice, versatile e preciso nel coltivare e stimolare le piccole popolazioni di cellule, e recuperando lisati per le analisi molecolari. La piattaforma è adatto a studi che impiegano cellule preziosi e / o reagenti.

Video: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Ricerca

Bioingegneria

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: una piattaforma automatizzata per la scoperta degli enzimi biomassa e ottimizzazione Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Una piattaforma Novel Stretching per applicazioni in cellule e tessuti mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered alginato costrutti: Una piattaforma per la cooperazione culturale delle popolazioni cellulari eterogenee

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Una piattaforma cellulare microarray high-throughput per l&#39;analisi correlativa cellulare Differenziazione e forze di trazione

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un metodo versatile di patterning proteine ​​e cellule

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubi di DNA come un Versatile strumento per lo studio di polimeri semiflessibili

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una piattaforma microfluidica multistrato per la conduzione di un'espressione genica prolungata senza cellule

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...