Name: easy and accurate mechano-profiling on micropost arrays
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Description: Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

This work was supported by the Committee Technology and Innovation (CTI) Switzerland grant 14796-PFLS-LSCTI. MicroPost arrays were generously provided by MicroDuits GmbH.

1. het kweken van cellen op MicroPOST Arrays Opmerking: Alle stappen moeten in een bioveiligheid kast worden uitgevoerd om de steriliteit te waarborgen. De hier gegeven volumes zijn voor T25 celcultuur flessen. Het celkweekmedium recept en cellen zaaidichtheid zijn geoptimaliseerd voor botkanker cellijnen HuO9 en M132. <ol><li> Bereid een celkweek voor het zaaien op een MicroPOST array. <ol><li> Inspecteer de celkweek kwaliteit door het plaatsen van de kolf op een standaard lichtmicroscoop...

<ol>
	<li>Sharma, R. I., Snedeker, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+biomechanical+gradients+for+directed+bone+marrow+stromal+cell+differentiation+toward+tendon+and+bone.">Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone.</a> Biomaterials. 31 (30), 7695-7704 (2010).</li><li>Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+biophysics+of+cancer+cells.">Biomechanics and biophysics of cancer cells.</a> Acta Biomater. 3 (4), 413-438 (2007).</li><li>Bartalena, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+assessing+adherent+single-cell+stiffness+in+tension:+design+and+testing+of+a+substrate-based+live+cell+functional+imaging+device.">A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device.</a> Biomed Microdevices. 13 (2), 291-301 (2011).</li><li>Tan, J. L., 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+lying+on+a+bed+of+microneedles:+An+approach+to+isolate+mechanical+force.">Cells lying on a bed of microneedles: An approach to isolate mechanical force.</a> PNAS. 100 (4), 1484-1489 (2003).</li><li>Akiyama, Y., Hoshino, T., Iwabuchi, K., Morishima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room+Temperature+Operable+Autonomously+Moving+Bio-Microrobot+Powered+by+Insect+Dorsal+Vessel+Tissue.">Room Temperature Operable Autonomously Moving Bio-Microrobot Powered by Insect Dorsal Vessel Tissue.</a> PloS ONE. 7 (7), (2012).</li><li>Biais, N., Ladoux, B., Higashi, D., So, M., 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=Cooperative+retraction+of+bundled+type+IV+pili+enables+nanonewton+force+generation.">Cooperative retraction of bundled type IV pili enables nanonewton force generation.</a> Plos Biology. 6 (4), 907-913 (2008).</li><li>Wuang, S. C., Ladoux, B., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Chemo-Mechanical+Effects+of+an+Anti-Cancer+Drug+Emodin+on+Breast+Cancer+Cells.">Probing the Chemo-Mechanical Effects of an Anti-Cancer Drug Emodin on Breast Cancer Cells.</a> Cell Mol Bioeng. 4 (3), 466-475 (2011).</li><li>Schoen, I., Hu, W., Klotzsch, E., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Cellular+Traction+Forces+by+Micropillar+Arrays:+Contribution+of+Substrate+Warping+to+Pillar+Deflection.">Probing Cellular Traction Forces by Micropillar Arrays: Contribution of Substrate Warping to Pillar Deflection.</a> Nano Lett. 10 (5), 1823-1830 (2010).</li><li>Lemmon, C. 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=Shear+Force+at+the+Cell-Matrix+Interface:+Enhanced+Analysis+for+Microfabricated+Post+Array+Detectors.">Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors.</a> Mech Chem Biosyst. 2 (1), 1-16 (2005).</li><li>Lam, R. H. W., Weng, S. N., Lu, W., Fu, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+subcellular+measurement+of+cell+stiffness+using+a+microengineered+stretchable+micropost+array+membrane.">Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane.</a> Integr Biol-UK. 4 (10), 1289-1298 (2012).</li><li>Roure, O., 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=Force+mapping+in+epithelial+cell+migration.">Force mapping in epithelial cell migration.</a> PNAS. 102 (39), 14122-14122 (2005).</li><li>Papenburg, B. J., Rodrigues, E. D., Wessling, M., Stamatialis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+role+of+material+surface+topography+and+wettability+on+cell-material+interactions.">Insights into the role of material surface topography and wettability on cell-material interactions.</a> Soft Matter. 6 (18), 4377-4388 (2010).</li><li>Badique, 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=Directing+nuclear+deformation+on+micropillared+surfaces+by+substrate+geometry+and+cytoskeleton+organization.">Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization.</a> Biomaterials. 34 (12), 2991-3001 (2013).</li><li>Sniadecki, N. 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=Magnetic+microposts+as+an+approach+to+apply+forces+to+living+cells.">Magnetic microposts as an approach to apply forces to living cells.</a> PNAS. 104 (37), 14553-14558 (2007).</li><li>Weng, R. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silicone-based+stretchable+micropost+array+membrane+for+monitoring+live-cell+subcellular+cytoskeletal+response.">A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response.</a> Lab Chip. 12, 731-740 (2012).</li><li>Desai, R. A., Yang, M. T., Sniadecki, N. J., Legant, W. R., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Post-Array-Detectors+(mPADs):+an+Approach+to+Isolate+Mechanical+Forces.">Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces.</a> J Vis Exp. (8), e311 (2007).</li><li>Yang, M. T., Fu, J. P., Wang, Y. K., Desai, R. A., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+stem+cell+mechanobiology+on+microfabricated+elastomeric+substrates+with+geometrically+modulated+rigidity.">Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity.</a> Nat Protoc. 6 (2), 187-213 (2011).</li><li>Sniadecki, N. J., Han, S. J., Ting, L. H., Feghhi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+in+Cell+Biology.">Micropatterning in Cell Biology.</a> Methods in Cell Biol. 121, 61-73 (2014).</li></ol>

Dit werk tracht het gebied van trekkracht microscopie vooraf door substantieel verlagen van technische en praktische belemmeringen voor de toegang. Deze belemmeringen van twee kanten komen. Eerst en vooral zijn de talloze niet-triviale technische uitdagingen die moeten worden overwonnen om reproduceerbaar vervaardigen en experimenteel Commissie MicroPOST array. Ten tweede, is het eveneens niet-triviale behoefte aan een betrouwbare semi-automatische analyse van cel krachten - rekening houdend dat een typisch experiment d...

Mechanisch gevoelige cellen gebaseerde assays mogelijk maken onderzoeken hechtende cellen met een breed toepassingsgebied dat de centrale rol kunnen spelen in de mechanica celbiologie weerspiegelen. Deze toepassingen richten zich vaak op de onderliggende mechanismen die subcellulaire processen of whole-cell gedrag sturen. Enerzijds kunnen externe omgevingsfactoren zoals extracellulaire matrix samenstelling of matrix stijfheid dramatische invloed op de mechanische en biologische reactie van een cel. 1 Hetzelfde kan worden waargenomen na toepassing van vele klassen van farmaceutisch werkzame verbindingen, waarvan de gevolgen zijn vaak gekenmerkt gebruik celkweekmodellen. 2 Anderzijds genotypische eigenschappen, zoals die veroorzaakt door spontane of experimenteel geïnduceerde genetische mutaties, kunnen duidelijke veranderingen in cel fenotype die zijn geassocieerd met veranderingen in het cytoskelet structuur en functie induceren. 3 Deze voorbeelden zijn slechts enkele van de vele mogelijkeOnderwerpen waarvoor mechanische fenotypering van cellen is relevant, en al deze zijn nuttig onderzocht met MicroPOST arrays. Op het moment van dit schrijven, hebben ongeveer 200 artikelen gepubliceerd beschrijven cel-MicroPOST interactie. Deze werken bespreken theoretische aspecten van MicroPOST afbuiging principes en praktische instructies voor de vervaardiging ervan. Het eerste artikel beschrijft de interactie van cellen en flexibele MicroPOST arrays werd gepubliceerd door Tan en collega&#39;s in 2003. 4 In tegenstelling tot de klassieke trekkracht microscopie (TFM), waar continue zachte substraten worden gebruikt voor het schatten nanonewton schaal cel contractiliteit, Tan et al. beschrijft een werkwijze met meerdere dicht bij elkaar gelegen verticale balken van silicone elastomeer. De belangrijkste voordelen van deze techniek blijken uit twee belangrijke functies. Eerst teneinde de cel-substraat schijnbare stijfheid veranderen men hoeft alleen de MicroPOST afmetingen te veranderen terwijlde substraatsamenstelling overigens gelijkblijvende en aldus verschillen in oppervlakte topologie en chemie vermijden. Tweede micropalen handelen als individuele veren die discreet kan worden geanalyseerd met geweld en ruimtelijke resoluties over de volgorde van de afzonderlijke focale adhesies en kan de analytische uitdagingen die inherent zijn aan een soortgelijk onderzoek door standaard TFM zijn verminderen. Vandaag is de waaier van toepassingen voor MicroPOST arrays veel groter is dan alleen het in kaart brengen van de krachten voor een paar enkele cellen. Bijvoorbeeld, Akiyama meldt het gebruik van een geïsoleerde dorsale vaatweefsel uit mottenrupsband als een actuator voor een MicroPOST array teneinde een insect spier-powered autonome micro-robot ontwikkelen. 5 De meeste gepubliceerde aanvragen van micropalen gericht op studies van medische aandoeningen zoals infecties of kanker. Zo zijn MicroPOST arrays gebruikt om de kracht genereren van gebundelde type IV pili van Neisseria gonorrh bestuderenOEA kolonies die is gekoppeld aan signaalcascades verbeteren infectie. 6 Anderen hebben gebruikt om micropalen borstkankercellen behandeld met farmaceutische samenstellingen gericht op de cytoskelet bestuderen. 7 Afbuiging van een MicroPOST wordt vaak beschreven met behulp van klassieke bundel theorie voor een cantilever met een end load veronderstelling dat de cel hecht alleen het uiterste puntje van de MicroPOST. Hier is de uitgeoefende kracht F die een afbuiging δ veroorzaakt afhankelijk van de MicroPOST&#39;s &quot;buigstijfheid&quot; k en wordt berekend door: <img alt="Vergelijking 1" src="/files/ftp_upload/53350/53350eq1.jpg" /> (1) met E, I en L zijn de Young&#39;s modulus, oppervlakte traagheidsmoment en straallengte resp. Echter, de resultaten van deze vergelijking geven slechts een algemene benadering van de krachten op het werk sinds balk snijden en buigen als substraat kromtrekken niet in ac worden genomentellen. Gezien het feit dat micropalen zijn meestal gemaakt van zachte materialen zoals polydimethylsiloxaan (PDMS) -gebaseerde siliconenrubber deze factoren moeten worden opgenomen. . Schoen et al aangetoond dat er zo&#39;n correctiefactor op basis van de verhouding van de MicroPOST (L / D) en de Poisson verhouding v de overeenkomstige polymeer 8 wordt gegeven door.: <img alt="Vergelijking 2" src="/files/ftp_upload/53350/53350eq2.jpg" /> (2) Met T tilt (v) een kantelbare coëfficiënt die fitting parameter a = 1,3 zoals kan worden gevonden in hetzelfde artikel omvat: <img alt="Vergelijking 3" src="/files/ftp_upload/53350/53350eq3.jpg" /> (3) Dat betekent MicroPOST de gecorrigeerde stijfheid k corr is het product van de zuivere buigstijfheid k = k buigen en de correctiefactor corrgegeven door: <img alt="Vergelijking 4" src="/files/ftp_upload/53350/53350eq4.jpg" /> (4) Daarom moet cel kracht berekeningen worden uitgevoerd met de meer verfijnde variatie van vergelijking (1) leest nu: <img alt="Vergelijking 5" src="/files/ftp_upload/53350/53350eq5.jpg" /> (5) De impact van de correctie wordt meer duidelijk zodra typische waarden voor MicroPOST afmetingen worden gebruikt. Bijvoorbeeld, een 15-micron lang MicroPOST met een cirkelvormige dwarsdoorsnede en een diameter van 5 urn gemaakt van PDMS-gebaseerde siliconen rubber tot een correctiefactor van 0,77 en dus een gecorrigeerde berekening zou de uitgeoefende krachten cel overschatten met 23%. Dit wordt nog ernstiger voor micropalen met kleinere aspect ratio. Traditioneel is MicroPOST beeldanalyse ook gebaseerd op geïdealiseerde balk buigen theorie. In 2005 heeft de groep die het gebruik van de MICR pionieropost arrays publiceerde een beeldanalyse-software geschikt voor MicroPOST analyse 9 De software vereist een softwarelicentie en de gebruiker moet drie beelden voor elke positie in te nemen.; één elk van de MicroPOST de boven- en onderkant vliegtuigen in de transmissie-modus en een andere in fluorescentie mode met de gekleurde cel. Na vergelijking van de bovenste en onderste posities voor elke MicroPOST bepaalt de software een kracht vectorveld en berekent daarmee samenhangende parameters als kracht per paal. Andere software pakketten bestaan ​​en hun analyse principes zijn kort in de desbetreffende artikelen die ze beschrijven genoemd, maar deze analytische software pakketten zijn over het algemeen niet voor iedereen beschikbaar. 10,11 De MicroPOST matrices voor mapping cel krachten kunnen worden geclassificeerd als zijnde in een orthogonale MicroPOST layout of een hexagonaal is, waarvan de laatste het voordeel van gelijke afstand tussen alle spleten naaste micropalen. Typische micropalen have een cirkelvormige dwarsdoorsnede en de afmetingen variëren van 1,0 urn tot 10 urn in diameter en 2 tot 50 urn lengte. 4 echter micropalen met elliptische of vierkante doorsnede zijn ook gemeld. 12,13 Het gebruik van PDMS-gebaseerde siliconen mengsels MicroPOST materiaal maakt voor het toevoegen van nanodeeltjes in het mengsel. Bijvoorbeeld het toevoegen van kobalt nano-staven maakt een magnetische activering van de MicroPOST en dus geeft een andere vrijheidsgraad om potentiële experimentele ontwerpen. 14 De meeste groepen produceren hun MicroPOST arrays op vlakke harde ondergronden zoals dekking van glas of in een petrischaal. Echter, Mann en collega meldde onlangs een MicroPOST matrix gevormd op een uitrekbaar membraan. 15 Dit maakt de toepassing van cel strekkrachten hechtende cellen tijdens de studie levende cellen subcellulaire dynamische responsen qua cel contractiliteit. De alom gebruikte en meest Estabsteld werkwijze voor het maken MicroPOST arrays gebaseerd op zachte lithografie zoals beschreven in de protocollen van inzichtelijke Sniadecki en collega. 16-18 Kortom standaard cleanroom processen worden toegepast om de microstructuren op een siliciumwafer gebruikt SU8 fotoresist genereren. Daarna volgt een kopieerproces waarbij de siliconenrubber gegoten via structuren overzetten in mallen. In een tweede stap deze schimmels worden gebruikt om de oorspronkelijke microstructuur behulp siliconenrubber op een gekozen substraat repliceren. Maar ondanks de grote en groeiende aantal publicaties in verband met de toepassing ervan, de oprichting van een productieproces voor micropalen neemt aanzienlijke hoeveelheid tijd, zelfs voor micro-technische deskundigen; er zijn veel processtappen die optimalisatie en aanpassing aan de specifieke testomgeving en MicroPOST lay-out nodig om een ​​acceptabel kwaliteitsniveau opleveren. Commerciële MicroPOST arrays zijn nu beschikbaar in een kant-en-to-gebruik (&quot;off-the-shelf&quot;) formaat met een constante hoge kwaliteit. Als zodanig zijn ze een alternatief voor de complexe en tijdrovende productieproces vereist ter plaatse produceren. In dit artikel werd een commercieel verkrijgbaar MicroPOST serie gebruikt voor het in kaart brengen cellulaire krachten afbeelding één helder-veld microscopie gebruikt. Nog belangrijker is dit artikel beschrijft en documenteert een volledig functionele open-source software genaamd MechProfiler, die beschikbaar zijn voor download als aanvullend materiaal om dit manuscript is. Een actief gehandhaafd versie van de software is ook te vinden op http://www.orthobiomech.ethz.ch. De combinatie van een &quot;off-the-shelf&quot; test en een compatibele open-source analyse software aanzienlijk verlaagt de ingang hindernis om nauwkeurige TFM experimenten te bereiken. Onderzoekers zonder toegang tot ofwel clean room faciliteiten of de ontwikkeling van software ervaring met succes kan analyseren cellulaire krachten. Het stelt een gebruiker om zich te concentreren op het mechanosensitivity assay uitgang in plaats van de technologie zelf, en maakt trekkracht metingen beschikbaar om een ​​bredere gemeenschap. Bovendien is dit een belangrijke stap op de weg naar volledig automatische screening van MicroPOST arrays effenen. De MechProfiler analyse software verwerkt afbeeldingen in bestandsformaat tiff, png, bmp en jpg. De beelden kunnen worden gemaakt met behulp van fluorescentie, fase contrast of fel-veld lichtmicroscopie. Het standalone programma loopt samen met de vrije Matlab Compiler Runtime (verkrijgbaar bij: Figuur 12) en de onderliggende algoritmes mogelijk maken gestroomlijnde beeldverwerking, waarmee de gebruiker om beelden met één of meerdere cellen te verwerken ongeveer 1 min. Verder kunnen deze cellen ofwel levend of &quot;vast&quot;. De MechProfiler software kan aanzienlijk verhogen gegevensanalyse throughput beroept op reproduceerbaarheid van de kwaliteit commerciële MicroPOST arrays, meer bepaald de standaard &amp; #8220, niet afgebogen &quot;-positie van elk record in de array kan worden aangenomen tegenover een ideale rooster (fabricageafwijkingen voor het raster in de arrays gebruikt voor deze studie minder dan 100 nm). Kortom men opent een selectie van beeldbestanden voor analyse, gewassen ze naar de regio van belang, bepaalt de ambten waarop cellen of die moeten worden weggegooid, bepaalt de post posities, berekent de doorbuiging / krachten tegen de ideale raster, en ten slotte slaat alle cel-specifieke gegevens met de mogelijkheid voor de export, met inbegrip van een standaard kantoor spreadsheet.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>T25 cell culture flasks</td>
			<td>Nunc</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS-buffer</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium DMEM/F12</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS South America</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100x</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge vials</td>
			<td>Sarstedt</td>
			<td>62.554.502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropost array&#160;</td>
			<td rowspan="2">MicroDuits</td>
			<td rowspan="2">MPA-col1/FN</td>
			<td>Micropost dimensions:</td>
		</tr>
		<tr>
			<td>pre-coated with Collagen I/Fibronectin</td>
			<td>&#216;= 6.4 &#181;m, l=18.2 &#181;m; grid=&#160;13 &#181;m, kcorr= 2.87 nN/&#181;m</td>
		</tr>
		<tr>
			<td>Ethanol abs p.A.</td>
			<td>Merck</td>
			<td>100.983</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Nunc</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered 10%</td>
			<td>Sigma</td>
			<td>HT5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Blue G-250</td>
			<td>Sigma</td>
			<td>27815</td>
			<td>Coomassie blue</td>
		</tr>
		<tr>
			<td>Methanol ACS p.A.</td>
			<td>Merck</td>
			<td>1.06009</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>WillCo Wells BV</td>
			<td>GWSb-3522</td>
			<td>35 mm diameter, aperture 22&#160;mm</td>
		</tr>
		<tr>
			<td>T-100 Eclipse</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>D3-L3</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Camera controler</td>
		</tr>
		<tr>
			<td>DS Fi2</td>
			<td>Nikon</td>
			<td></td>
			<td>Camera</td>
		</tr>
	</tbody>
</table>

easy and accurate mechano-profiling on micropost arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Het belangrijkste voordeel van de beschreven techniek ligt in de eenvoud en de mogelijkheden voor snelle en effectieve integratie in routine laboratoriumwerk. De combinatie van commerciële sensor arrays van hoge kwaliteit gecombineerd met open-source software geeft informatie over mechanisch-gevoeligheid die anders toegang tot de cleanroom faciliteiten en diepgaande kennis van de beeldanalyse en de ontwikkeling van software zou nodig. Figuur 1 toont de workflow van de gepresenteerde methode. Het begint...

Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

Easy and Accurate Mechano-profiling on Micropost Arrays

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Gemakkelijk en nauwkeurig voor mechanische profilering op MicroPOST Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

easy-and-accurate-mechano-profiling-on-micropost-arrays

Research

JoVE Journal

Bioengineering

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Studeren Cell Rolling Trajecten op Asymmetrische Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Hoog rendement, Site-specific Transfectie van adherente cellen met siRNA Met behulp van micro-elektrode arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Afsluitbare Femtoliter Kamer Arrays voor Celvrije Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Ligand Nano-cluster Arrays in een ondersteunde lipide dubbellaag

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Interfacing Microfluidics met micro-elektrode Arrays voor het bestuderen van de neuronale communicatie- en axonale signaal vermeerdering

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Humane iPSC-afgeleide cardiomyocyte netwerken op multi well micro-elektrode arrays voor terugkerende actie potentiële opnames

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Fabricage van Ti3C2 MXene Microelectrode Arrays voor In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

Substructuur Analyzer: Een gebruiksvriendelijke workflow voor snelle verkenning en nauwkeurige analyse van cellulaire lichamen in fluorescentiemicroscopie beelden

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

Flowcytometrie met hoge dimensionaliteit voor analyse van de immuunfunctie van ontlede implantaatweefsels

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Fabricage van 3D cardiale microtissue arrays met behulp van menselijke iPSC-afgeleide cardiomyocyten, cardiale fibroblasten en endotheelcellen

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...