Gli autori desiderano ringraziare che Fedex Institute of Technology presso l&#39;Università di Memphis per il loro sostegno. Gli autori desiderano ringraziare gli studenti del gruppo di progetto di design di alto livello presso il Dipartimento di Ingegneria Meccanica presso l&#39;Università di Memphis (David Butler, Jackie Carter, Dominick Cleveland, Jacob Shaffer), Daniel Kohn del dipartimento Università di Memphis Engineering Technology per il controllo motore , e il dottor Bin Teng e la signora Charlean Luellen per il loro aiuto in coltura cellulare. Questo lavoro è stato sostenuto da K01 HL120912 (ER) e R01 HL123540 (CMW).

1. Costruzione di membrana con Well Mura per la conservazione di cellule di coltura (vedi Figura 1D per il prodotto finale) <ol><li> Utilizzando polidimetilsilossano (PDMS) fogli rivestiti con collagene I, tagliare il contorno della membrana flessibile con un bisturi o un dado. </li><li> Posizionare ogni membrana in una piastra di Petri di 60 mm per immagazzinaggio. </li><li> Creazione di pareti: <ol><li> Mescolare PDMS in un rapporto di 10: 1 in peso di elastomero A a elastomero B (induritore). </li><li> Versare 5 ml di PDMS completamente misti in provette da 50 ml. </li><li> Mettere 50 ml tubi con PDMS TDS orizzontalmente in un fornetto. </li><li> Utilizzare la funzione del rotore per rivestire le pareti interne dei tubi a 8 rpm durante il tempo di polimerizzazione. A RT, i PDMS saranno perfettamente polimerizzato in 2 giorni. </li><li> Rimuovere il PDMS dal tubo in una cappa sterile coltura cellulare. </li><li> Utilizzando una nuova lama di rasoio, partizionare il cilindro di PDMS in sezioni 4 millimetri di altezza. </li><li> Posizionare le sezioni, che servirà come il wa membranalls, in un contenitore piatto Petri senza consentire alle pareti diventino creased. </li></ol></li><li> Selezionare e centrare una parete di PDMS su una membrana, mantenendo i due nella scatola di Petri. </li><li> Posizionare delicatamente PDMS TDS (10: 1 ratio) sul perimetro esterno della parete da utilizzare come collante. Evitare la formazione di spazi vuoti tra la parete e la membrana essendo lì per trattenere liquidi. </li><li> Inserire ogni piatto Petri contenente la membrana completato e coperto in stufa a 70 ° C per 24 ore per curare. </li></ol> 2. Correlazione di giri del motore con morsetto Spostamento o crescita radiale per la calibrazione (Figura 2) <ol><li> Avviare il software di controllo del motore. </li><li> Spostare i morsetti del dispositivo utilizzando l&#39;impostazione manuale del software del motore. </li><li> Misurare la distanza e registrare la posizione di conteggio del motore tra opposte morsetti sia cilindrata minima e massima delle pinze. </li><li> Calcolare la variazione percentuale distance fra le ganasce in funzione del conteggio motore (~ 0-75k conteggi). Ciò indica la deformazione massima potenziale raggiunto sulla membrana. </li></ol> 3. Applicazione di Stretch su Cell Line polmone mouse epiteliale (MLE12) <ol><li> Seed MLE12 cellule a 2,5 milioni di cellule sulla membrana flessibile (Fase 1) per pozzetto da confluenti entro due giorni. La densità di semina può variare per diversi tipi di cellule. </li><li> Assicurarsi che l&#39;attuatore meccanico è in una posizione completamente rilassato. <ol><li> Rimuovere cellule di coltura. </li><li> Utilizzando 1,5 millimetri punzoni biopsia, perforare due fori in ciascuna delle sei morsetto (vedi Figura 1D) linguette della membrana. Il posizionamento radiale dei fori determina la quantità di pre-tensionamento esperienze membrana inizialmente. Perforare i fori ad un raggio di 20,5 mm sulle linguette membrana se non pretensionamento è desiderato. </li><li> Posizionare la membrana sulla barella con i fori punzonati allineando i perni nei morsetti.Primo posto morsetti a posto. Serrare le viti una alla volta alternando i lati. </li><li> Aggiungere 1 ml terreni di coltura delle cellule. </li></ol></li><li> Posizionare il dispositivo sul palco microscopio centraggio mezzo della membrana con il percorso ottico. </li><li> Utilizzare nastro o magneti (se possibile) per fissare il dispositivo alla fase. Una volta fissato, il controllo in-plane (parallela alla membrana) e verticale (direzione z) del dispositivo meccanico con il controllore del microscopio. </li><li> Applicare stirata con il software fornito dal produttore controllando manualmente la posizione e la velocità di rotazione del motore 20. </li></ol> 4. Misura di mitocondriali specie reattive dell&#39;ossigeno (ROS) <ol><li> Una volta che le cellule sono confluenti, aggiungere 1-2 ml di RT DMEM con indicatore superossido mitocondriale (5 mM concentrazione finale) direttamente sulle cellule. </li><li> Incubare per 10 minuti a 37 ° C. </li><li> Lavare le cellule delicatamente per tre volte con tampone riscaldato a bagnomaria per37 ° C. </li><li> Porre immediatamente la membrana flessibile sulla barella (Step 2.2) già in atto con un microscopio confocale in posizione verticale. </li><li> Aggiungere 1 ml di DMEM con terreno privo di fenolo-rosso e 25 mm di HEPES. </li><li> Impostare filtri di eccitazione / emissione di 510/580 nm. </li><li> Immagine più campi ogni 15 minuti per creare il decorso temporale desiderato di produzione di superossido mitocondriale. </li><li> Dalle immagini catturate, intensità della fluorescenza registrare istogrammi ad ogni intervallo di tempo utilizzando il software in grado di quantificare l&#39;intensità di fluorescenza. </li></ol> 5. Applicazione di Stretch e microscopia a forza atomica (AFM) NOTA: Questi passaggi sono previsti per un AFM combinazione specifica microscopio ottico (figura 4 e Materiali List). <ol><li> Preparare la AFM per l&#39;esperimento. <ol><li> Aumentare l&#39;altezza della testa AFM alla sua posizione massima nella direzione z. </li><li> Mettere estensori sulle gambe della AFM persollevare il piano a cui sbalzo contatti AFM il campione. Il campione e la testa AFM deve essere sollevato per ospitare l&#39;altezza del dispositivo meccanico. </li></ol></li><li> Preparare il microscopio ottico (se disponibile). <ol><li> Rimuovere la piastra di scansione AFM. Rimuovere l&#39;obiettivo desiderato. </li><li> Aggiungi un distanziatore all&#39;obiettivo. L&#39;altezza del distanziatore dipenderebbe l&#39;obiettivo e l&#39;AFM specifico set-up, ma è necessario se imaging ottico è desiderata dal piano di osservazione sarà spostamento nella direzione z di una quantità pari alla barella e altezza adattatore (Figura 5A). Si noti che la AFM fornisce solitamente basso ingrandimento delle immagini da un percorso ottico sul dispositivo. </li><li> Montare la parte posteriore obiettivo desiderato nella sua posizione. Posizionare lo scanner al AFM. </li><li> Avviare il software AFM. Avviare tutte le sorgenti luminose necessari tra cui la sorgente luminosa per misure di fluorescenza. </li><li> Montare un chip con una trave a sbalzo cheè opportuno che le misure desiderate. 200 pN / rigidità nm o inferiore è preferibile quando si misura il modulo elastico di cellule vive. </li><li> Allineare il laser e calibrare la rigidità sbalzo secondo le indicazioni del fabbricante su un vetrino di vetro montato sul dispositivo. </li></ol></li><li> Preparare una membrana, come nella fase 1, ma con le seguenti modifiche. <ol><li> Immediatamente prima di montare la membrana al dispositivo tenditore, tagliare le pareti di circa 1 mm di altezza. Questo impedisce l&#39;interferenza sperimentato tra le pareti della membrana e la cella di carico. </li><li> Montare la membrana sul dispositivo meccanico come descritto 3.2.1-3.2.3. </li><li> Rimuovere terreni di coltura cellulare per evitare danni allo scanner AFM in caso di fuoriuscita. </li><li> Coppia il dispositivo con un adattatore (Figura 5A). Posizionare il dispositivo meccanico con l&#39;adattatore sullo scanner. </li><li> Allungare la membrana per il livello di deformazione a trazione desiderato. </li></ol></li><li&gt; Nano-indentazione di cellule allungate. <ol><li> Aggiungere un numero limitato (&lt;0,5 ml) il volume dei media sulle cellule per evitare scanner AFM o danni microscopio a causa di una caduta. </li><li> Engage trave a sbalzo con la membrana. </li><li> Seguire il protocollo del particolare dispositivo AFM per acquisire aree di interesse. </li></ol></li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

Un dispositivo unico per l&#39;imaging cellulare dal vivo durante allungamento meccanico è stato sviluppato; e questo dispositivo è stato utilizzato in un protocollo per studiare epiteliali polmonari mechanobiology cellule. In studi preliminari, si è constatato che un singolo tratto tenuta stimolato la produzione di superossido mitocondriale nelle cellule epiteliali bronchiali. Inoltre, è stato dimostrato che un aumento dei livelli di sollecitazioni meccaniche causate danno diretto per l&#39;integrità di un monostr...

Le cellule sono sottoposte a sollecitazioni meccaniche in molti tessuti, e questa stimolazione meccanica ha dimostrato di promuovere cambiamenti nei modelli di espressione genica, il rilascio di fattori di crescita, citochine, o rimodellamento della matrice extracellulare e citoscheletro 1-4. I segnali intracellulari trasdotte da tali stimoli meccanici avvengono attraverso il processo di meccanotrasduzione 5-7. Nel sistema respiratorio, un risultato di mechanotransduction è l&#39;aumento delle specie reattive dell&#39;ossigeno (ROS) 8,9 e citochine pro-infiammatorie 10 in cellule epiteliali polmonari in presenza di deformazione a trazione ciclica. Forte evidenza suggerisce anche che un&#39;eccessiva deformazione a trazione porta a dirigere ferita all&#39;epitelio alveolare, oltre alle risposte biochimiche delle cellule 11-14. Anche se il fuoco qui è soprattutto sulla risposta delle cellule polmonari a deformazione meccanica, percorsi indotte da meccanotrasduzione svolgono un ruolo chiave nei basic funzione di molti tessuti del corpo umano, compresa la regolazione del tono vascolare 15 e lo sviluppo della piastra di crescita 16. Il crescente interesse meccanotrasduzione ha portato allo sviluppo di numerosi dispositivi per l&#39;applicazione di fisiologicamente rilevanti sollecitazioni meccaniche alle cellule in coltura e tessuti. In particolare, i dispositivi che applicano lo sforzo a trazione, che è una forma comune di carico meccanico sperimentato da tessuto, sono popolari 11,17-19. Tuttavia, molti dei dispositivi disponibili sono sia concepita come un bioreattore per applicazioni di ingegneria tissutale o non sono favorevoli a delle immagini in tempo reale con la stirata. In quanto tale, vi è la necessità di sviluppare strumenti e metodi in grado di visualizzare le cellule e tessuti in tensione per facilitare la ricerca di percorsi di meccanotrasduzione. Qui, un dispositivo che si estende in piano meccanico è stato progettato e sono stati sviluppati protocolli per applicare mforme ultiple di tensione ai tessuti e cellule pur consentendo l&#39;imaging delle risposte biochimiche e meccaniche in tempo reale (Figura 1A-D). Il dispositivo utilizza sei morsetti equidistanti disposte circonferenzialmente ad afferrare una membrana flessibile e applicare una in piano, radiale distensione fino a circa il 20% (Figura 1B). Il dispositivo di azionamento può essere collocato in un incubatore di coltura cellulare per un periodo di tempo prolungato, mentre il motore (Figura 1C) è posizionato all&#39;esterno dell&#39;incubatrice e controllato da software proprietario fornito dal fornitore del motore. Il motore è collegato ad un driver lineare, che fa ruotare una camma interna, guidando i sei morsetti barella uniformemente in tensione e rilassamento. Oltre al dispositivo meccanico, membrane flessibili personalizzati sono stati creati da commercialmente disponibili coltura cellulare membrane pronti per essere utilizzati nel sistema meccanico. Poi pareti circolari (con un diametro di circa28 mm) sono state fatte e attaccato alla membrana flessibile in modo che le cellule possono essere coltivate solo in questa regione del profilo ceppo ben descritto. Al fine di determinare se il posizionamento di queste membrane all&#39;interno del dispositivo di azionamento fornirebbe una sollecitazione uniforme e isotropo nel centro della membrana flessibile, analisi agli elementi finiti è stata condotta utilizzando software disponibile in commercio (Figura 1E-F). La membrana flessibile è stato modellato con condizioni al contorno simmetriche e utilizzando tutti gli elementi quadrangolari per la maglia. Gli anelli concentrici visto nella trama di contorno di massima deformazione principale mostrato nella Figura 1F indicano la distribuzione isotropa del ceppo. Il ceppo sperimentato dalla membrana è stata misurata registrando immagini marcature attraverso carico (Figura 2). Figura 2D mostra che la deformazione media membrana misurata in direzioni radiali e assiali era approssimativamente linearerispetto al motore applicata conta fino a un ceppo lineare massima di 20%. Non c&#39;era alcuna differenza significativa tra i livelli di deformazione misurati durante distensione rispetto a quelli misurati durante la retrazione nella posizione di riposo. Successivamente, lo spostamento di cellule umane bronchiali epiteliali (16HBE) e loro nuclei coltivate sulla membrana flessibile personalizzato sono stati misurati. Fluorescente (DAPI) nuclei delle cellule 16HBE sono stati ripresi utilizzando un obiettivo 20X al microscopio confocale, mentre lo spostamento a cellula intera è stata misurata con le immagini registrate contrasto di fase con un microscopio digitale. Come si vede in figura 3, la deformazione misurato dallo spostamento dei nuclei era simile a quello misurato per spostamento di marcature sulla membrana, fino a ~ 20% di deformazione lineare. Ciò conferma che il ceppo applicato alle membrane è stata trasmessa alle cellule aderenti. I protocolli che descrivono l&#39;uso del dispositivo personalizzato su un microscopio tradizionale e una forza microscop atomicae sono previste nei seguenti passi.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SmartMotor NEMA 34: 3400 Series</td>
			<td>MOOG Animatics</td>
			<td>SM3416D</td>
			<td>Integrated motor, controller, amplifier, encoder and communications bus</td>
		</tr>
		<tr>
			<td>Flexcell Membrane (Collagen I coated)</td>
			<td>Flexcell International Corp</td>
			<td>SM2-1010C</td>
			<td>3.5x5.25x0.020&#34;</td>
		</tr>
		<tr>
			<td>Sylgard 184&#160;</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>10:1</td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H1399</td>
			<td>DAPI stain</td>
		</tr>
		<tr>
			<td>MitoSOX</td>
			<td>Sigma-Aldrich</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiron</td>
			<td>Sigma-Aldrich</td>
			<td>D7389&#160;</td>
			<td>mitochondrial superoxide label</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
			<td>superoxide inhibitor</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-19</td>
			<td>Any centriguge tube can be used to create an area for imaging.</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Bellco Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLE12 Cells</td>
			<td>ATCC</td>
			<td>CRL-2110</td>
			<td>Mouse Lung Epithelial Cells&#160;</td>
		</tr>
		<tr>
			<td>16HBE cells</td>
			<td>ATCC</td>
			<td>CRL-2741</td>
			<td>Human Bronchial Epithelial Cells</td>
		</tr>
		<tr>
			<td>AFM Indentation Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever Beams for Nano-indentation</td>
			<td>Budget Sensors</td>
			<td>Si-Ni30</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM&#160;</td>
			<td>Asylum Research</td>
			<td>MFP3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td>Inverted microscope with 20X and 40X objectives.</td>
		</tr>
		<tr>
			<td>AFM Leg Extenders</td>
			<td>Asylum Research</td>
			<td>Not available</td>
			<td>AFM microscope</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite Element Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>Simulia</td>
			<td>6.12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Life Technologies</td>
			<td>EVOS XL Core</td>
			<td>Initially a self standing company, now owned by Life Technologies.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>2-photon upright microscope</td>
		</tr>
	</tbody>
</table>

live cell imaging during mechanical stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

Specie reattive dell&#39;ossigeno e Deformazione Precedenti studi hanno mostrato un aumento delle specie reattive dell&#39;ossigeno (ROS) nelle vie respiratorie e cellule epiteliali alveolari in risposta al tratto ciclica 21. Le specie reattive dell&#39;ossigeno includono molecole e radicali liberi derivati ​​da ossigeno molecolare con alta reattività di lipidi, proteine, polisaccaridi e acidi nucleici 22-24. ROS servire come un segnale comune intracellula...

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Imaging Live Cell durante meccanica Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

live-cell-imaging-during-mechanical-stretch

Research

JoVE Journal

Bioengineering

10.3K Views.  University of Tennessee Health Science Center. A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy. 

Video: Live Cell Imaging during Mechanical Stretch

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon crescita Stretch: Il meccanotrasduzione di crescita neuronale

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

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Bioingegneria

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Live-cellula Imaging di migrazione delle cellule che esprimono proteine ​​fluorescenti-taggato in una matrice tridimensionale

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Monitoraggio dei Mechanics parete durante la distribuzione stent in un recipiente

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Una tecnica Microfluidic per sondare deformabilità cellulare

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Da Veloce imaging di fluorescenza di Molecular legge Diffusione su membrane cellulare dal vivo in un microscopio commerciale

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Dispositivo robotico isokinetico per migliorare l'affidabilità di Test-Retest e Inter-Rater per misurazioni Stretch Reflex nei pazienti colpiti da corsa con spasticità

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Un sistema microfisiologico per studiare l'interazione leucocitaria-cellula endoteliale durante l'infiammazione

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...