Questo lavoro è stato fondato da Institut Curie, Parigi, Francia. La barella meccanico è stato progettato da Damien Cuvelier (Institut Curie) ed è prodotto da GREM (mecanique-grem.com). Il patterning su PDMS è stato sviluppato da Ammar Azioune (Bordeaux II University).

1. Passivazione del PDMS <ol><li> Tagliare un pezzo di PDMS circa 35 mm x 20 mm da una lamiera pre fatta (per esempio, GelPak, come elencato nella tabella dei materiali). </li><li> Rimuovere la parte superiore e gli strati protettivi peggiori di plastica (se necessario) e usare pinzette per posizionare i PDMS in plastica (non COLTURA CELLULARE trattati) piastra Petri. </li><li> Lavare le PDMS con etanolo al 70% per 5 minuti su un agitatore a 30 oscillazioni / min. </li><li> Asciugare la superficie scorre aria su di esso. </li><li> Illuminare con profondo UV (λ = 180 nm) per 5 minuti a una distanza dalle lampade UV di circa 5 cm (vedi scheda Materiali per riferimento lampada; parametri variano per lampade diverse). </li><li> Preparare 200 ml di soluzione di EDC / NHS per ogni pezzo PDMS (deve essere preparata poco prima dell&#39;uso perché la reattività della soluzione decade in pochi hr). Per 1 ml di 0,05 M MES + 0,5 tampone M NaCl a pH 6,0, aggiungere 11,5 mg di Sulfo-SSN e 19,2 mg di EDC. </li><li> Trasferire ilPDMS fogli dal capsula di Petri al coperchio della scatola di Petri, che non è stata illuminata. Questo farà sì che i dintorni di PDMS sono molto idrofobiche e facilitare il passaggio successivo. </li><li> Aggiungere la soluzione di EDC / NHS e incubare per 15 min a temperatura ambiente. </li><li> Sciacquare EDC / NHS con acqua. </li><li> Aggiungere la soluzione PLL-g-PEG (0,5 mg / ml in HEPES 10 mM pH 8,6) e incubare dal 3 ore durante la notte a temperatura ambiente. </li><li> Sciacquare il PLL-g-PEG con acqua. PDMS passivato (funzionalizzati con PLL-g-PEG) possono essere conservati per diversi giorni a 4 ° C. </li></ol> 2. Patterning del PDMS <ol><li> Prendete un foglio PDMS e posizionarlo su una fotomaschera quarzo sintetico recante la microfeatures per patterning (vedi Figura 1). Posizionare il lato PDMS recante il PLL-g-PEG di fronte al lato di cromo del fotomaschera. </li><li> Illuminare per 7 min attraverso la fotomaschera ad una distanza dalle lampade UV di circa 5 cm.</li><li> Aggiungere acqua sulla maschera + PDMS e sbucciare le PDMS lentamente la maschera. </li><li> Incubare con soluzione fibronectina a 50 mg / ml in tampone HEPES a pH 8,6 per 1 ora a temperatura ambiente. E &#39;possibile utilizzare altri proteine ​​ECM, ma questi non sono stati testati con questo protocollo. </li><li> Risciacquare con PBS. </li></ol> 3. Montaggio del dispositivo <ol><li> Montare i PDMS precedenza passivate sul dispositivo di stretching (vedi la discussione per la risoluzione dei problemi). <ol><li> Attaccare un lato delle PDMS alla parte fissa della barella. </li><li> Fissare l&#39;altro lato per la parte mobile della barella senza bloccaggio troppo del foglio PDMS. </li></ol></li><li> Tagliare un rettangolo di PDMS (22 mm x 19 mm) in una spessa PDMS stab e tagliare un altro rettangolo interno in modo da avere una piscina (o frame) che manterrà le cellule e supporti (vedere Figura 2). </li><li> Aggiungere grasso al silicone sotto questo PDMS piscina e posizionarlo sulla parte superiore del foglio PDMS per creare un medium mantenendo piscina. Il grasso permetterà lo scorrimento della piscina sopra il foglio PDMS nella filatura. </li></ol> 4. Patterning celle <ol><li> Staccare le cellule da una confluenza pallone di coltura 50% con Versene. </li><li> Contare le cellule e risospendere alla concentrazione di 200.000 cellule / ml. </li><li> Aggiungere 1 ml di sospensione cellulare in piscina (vedi la discussione per ulteriori dettagli). </li><li> Lasciate che le cellule si legano ai modelli per 10-30 min (a seconda del tipo di cellula, RPE-1 le cellule avranno 10 min e cellule HeLa 20 min). </li><li> Lavare delicatamente le cellule galleggianti con medio equilibrato (equilibrare medio in incubatrice). </li><li> Lasciate che le cellule sparse per qualche ora sui modelli (RPE-1 avrà bisogno di almeno 2 ore; cellule HeLa avranno bisogno di 3 ore). </li><li> Aggiungere un coprioggetto sulla parte superiore della piscina per evitare l&#39;evaporazione e medie perdite nel caso in cui PDMS pause. </li><li> Mettere il dispositivo su un microscopio invertito e iniziare imaging. Evitare l&#39;uso di immersione in olio objectives in quanto non funziona a causa di problemi di messa a fuoco. </li></ol> 5. Allungamento <ol><li> Ruotare la vite micrometrica correggendo la posizione di fase del x-, y-e z-asse (principalmente x). La posizione di fase deve essere corretta per contrastare l&#39;allargamento delle PDMS e la perdita di concentrazione. (Vedere &quot;Durante il tratto&quot; paragrafo nella discussione). </li></ol>

<ol>
	<li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).</li><li>Terenna, C. R., 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=Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast.">Physical mechanisms redirecting cell polarity and cell shape in fission yeast.</a> Curr. Biol. 18 (22), 1748-1753 (2008).</li><li>Guillot, C., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+epithelial+tissue+homeostasis+and+morphogenesis.">Mechanics of epithelial tissue homeostasis and morphogenesis.</a> Science. 340 (6137), 1185-1189 (2013).</li><li>Bosveld, F., 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=Mechanical+control+of+morphogenesis+by+Fat/Dachsous/Four-jointed+planar+cell+polarity+pathway.">Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.</a> Science. 336 (6082), 724-727 (2012).</li><li>Farhadifar, R., Roper, J. C., Aigouy, B., Eaton, S., Julicher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+cell+mechanics,+cell-cell+interactions,+and+proliferation+on+epithelial+packing.">The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.</a> Curr. Biol. 17, 2095-2104 (2007).</li><li>Rauzi, M., Verant, P., Lecuit, T., Lenne, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+anisotropy+of+cortical+forces+orienting+Drosophila+tissue+morphogenesis.">Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.</a> Nat. Cell Biol. 10, 1401-1410 (2008).</li><li>Mitrossilis, D., 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=Real-time+single-cell+response+to+stiffness.">Real-time single-cell response to stiffness.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., 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=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. Exch. , (2011).</li><li>Sinha, B., 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=Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae.">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li></ol>

Gli autori dichiarano interessi finanziari concorrenti.

Sebbene questa tecnica è stata utilizzata numerose volte e viene accuratamente testato, ci sono diversi passaggi critici che possono portare ad un esperimento fallito. Circa il PDMS: Per questo lavoro, GelPak, un foglio sottile PDMS disponibile in commercio, è stato utilizzato. In alternativa, i fogli di PDMS possono essere lanciate direttamente dalla miscela PDMS. Si consiglia di utilizzare GelPak perché è più riproducibile, ed è meno probabilità di rottura ...

Le cellule che compongono un tessuto in organismi superiori sono soggetti a tensioni meccaniche e forze traenti o provenienti dall&#39;ambiente esterno o da cellule circostanti 1,2. Cellule devono adattarsi e resistere a queste forze al fine di mantenere l&#39;integrità del tessuto. Tali forze sono importanti anche per i tessuti morfogenesi durante lo sviluppo 3,4. Applicando forze meccaniche su cellule coltivate è un modo di imitare ciò che potrebbe accadere in un tessuto, ma con un controllo quantitativo e indipendente di forma delle cellule e deformazione cella 5,6. Per questo, possono essere utilizzate diverse tecniche. Si può premere sulle cellule (cellula intera o parte di essa), ad esempio utilizzando AFM o derivati ​​7,8 o allungare il substrato le cellule crescono su. Il metodo descritto in questo documento dimostra come allungare un substrato piano placcato con cellule. Questa tecnica è stato originariamente sviluppato per valutare il ruolo delle forze esercitate su mcellule di mammifero itotic 9. Le cellule mitotiche rimanere connessi al substrato attraverso le fibre di retrazione e stretching membrana esercitata una forza su quelle fibre, che a loro volta hanno provocato la rotazione del fuso mitotico. L&#39;interesse di combinare microdisegnature adesivi e stretching è quello di raggiungere un controllo indipendente delle forze e forma delle singole celle. E &#39;per esempio possibile allungare una cella ovoidale in una forma rotonda perfettamente isotropa, mentre è applicata tratto monoassiale. Se le cellule non sono intrecciata su microdisegnature, stiramento uniassiale risultati in allungamento cellulare, con la maggior parte delle cellule avente un asse longitudinale allineato con l&#39;asse tratto. È poi difficile separare l&#39;effetto dell&#39;allineamento asse lungo e l&#39;effetto del tratto applicata alle cellule. Il dispositivo è adatto per qualsiasi imaging cellulare dal vivo, tra cui la microscopia a fluorescenza lasso di tempo, e la droga può essere aggiunto durante l&#39;esperimento. Il metodo UV micropatterning profondo 10è stato descritto in dettaglio nel Azioune et al. 11 patterning su PDMS è stata descritta in Azioune et al. 12 La presente allungamento protocollo è una versione video di Carpi et al. 13

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretching micropatterned cells on a pdms membrane

Forze meccaniche esercitate sulle cellule e / o tessuti svolgono un ruolo importante in numerosi processi. Abbiamo sviluppato un dispositivo di allungare le cellule placcato su un polidimetilsilossano (PDMS) membrana, compatibile con le immagini. Questa tecnica è riproducibile e versatile. La membrana PDMS può essere micropatterned al fine di limitare cellule o tessuti di una geometria specifica. Il primo passo è stampare microdisegnature sulla membrana PDMS con una tecnica UV profondo. La membrana PDMS è poi montato su un telaio meccanico. Una camera è vincolata in cima alla membrana con grasso biocompatibile per consentire scivolare durante l&#39;allungamento. Le cellule vengono seminate e ha permesso di diffondere per diverse ore sui microdisegnature. Il campione può essere allungato e non deformato più volte con l&#39;uso di una vite micrometrica. Ci vuole meno di un minuto per applicare il tratto per tutta la sua estensione (circa il 30%). La tecnica qui presentata non include un dispositivo motorizzato, che è necessario per unapplying cicli tratto ripetuti rapidamente e / o computer controllato stretching, ma questo può essere attuato. Stretching di cellule o di tessuto può essere di interesse per le questioni relative alle forze cellulari, risposta cellulare allo stress meccanico o morfogenesi dei tessuti. Questa presentazione video mostra come evitare i problemi tipici che potrebbero sorgere quando si fa questo tipo di apparentemente semplice esperimento.

<html> La tecnica presentata in questo protocollo video consentiva l&#39;applicazione di forze sulle fibre di retrazione di cellule di mammifero mitotiche. Infatti, durante la divisione cellulare, allo stadio mitotico, cellule di mammifero ritraggono a prendere la forma di una sfera e di lasciarsi alle spalle i cavi di actina sottili circondate da una membrana che sono attaccati al substrato. Questi cavi (fibre di retrazione), sono la memoria della geometria della cella prima di andare in divisione. Fare microdisegnature...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

Questo manoscritto presenta una tecnica per applicare o liberare forze su cellule o tessuti aderenti usando allungamento unidirezionale.

Stretching celle micropatterned su una membrana PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

stretching-micropatterned-cells-on-a-pdms-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

15.2K Views.  Institut Curie.  Questo manoscritto presenta una tecnica per applicare o liberare forze su cellule o tessuti aderenti usando allungamento unidirezionale. 

Video: Stretching Micropatterned Cells on a PDMS Membrane

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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

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

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

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

Ricerca

Bioingegneria

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Biologia dello sviluppo

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 ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Modellazione di microrganismi e microparticelle attraverso l'assemblaggio sequenziale assistito da capillarità

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Ingegneria

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 ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Preparazione di microambienti a matrice extracellulare tunable per valutare schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Generazione di modelli per microscopia a trazione micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Stretching celle micropatterned su una membrana PDMS

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Capitoli in questo video

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

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Una piattaforma Novel Stretching per applicazioni in cellule e tessuti mechanobiology

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

Modellazione di microrganismi e microparticelle attraverso l'assemblaggio sequenziale assistito da capillarità

Un metodo versatile di patterning proteine e cellule

Preparazione di microambienti a matrice extracellulare tunable per valutare schwann Cell Phenotype Specification

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

Generazione di modelli per microscopia a trazione micropattern

Generazione di microambienti cellulari multicue mediante fotopatterning UV di substrati tridimensionali di colture cellulari

Stretching celle micropatterned su una membrana PDMS

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Stencil micropatterning di cellule staminali umane pluripotenti per Probing spaziale Organizzazione di differenziazione Fates

Una piattaforma Novel Stretching per applicazioni in cellule e tessuti mechanobiology

Espansione nanostrutturati Substrati utilizzando la tecnica del punto per Nanotopographical Modulazione di Cell Comportamento

Modellazione di microrganismi e microparticelle attraverso l'assemblaggio sequenziale assistito da capillarità

Un metodo versatile di patterning proteine ​​e cellule

Preparazione di microambienti a matrice extracellulare tunable per valutare schwann Cell Phenotype Specification

Controllo della geometria cellulare attraverso il micropatterning laser assistito a infrarossi

Generazione di modelli per microscopia a trazione micropattern

Generazione di microambienti cellulari multicue mediante fotopatterning UV di substrati tridimensionali di colture cellulari

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

Un metodo versatile di patterning proteine e cellule