Name: easy and accurate mechano-profiling on micropost arrays
Uploaded: 2015-11-17T15:00:00
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. odling av celler på Micropost Arrays Obs: Alla steg måste utföras i en biosäkerhet skåp för att säkerställa sterilitet. De volymer som anges här är för T25 cellodlingsflaskor. Cellodlingsmediet recept och cellsåddtäthet är optimerade för ben cancercellinjer HuO9 och M132. <ol><li> Förbered en cellkultur för sådd på en micropost array. <ol><li> Inspektera cellodlingskvalitet genom att placera odlingskolven på en standardljusmikroskop. Se till att cellerna visar en typ...

<ol>
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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>

Detta arbete syftar till att främja det gäller dragkraft mikroskopi genom att väsentligt sänka tekniska och praktiska hinder för inträde. Dessa hinder kommer från två sidor. Först och främst är de många icke-triviala tekniska utmaningar som måste övervinnas för att reproducerbart tillverka och experimentellt beställa en micropost array. För det andra, är det likaledes icke-triviala behov av en tillförlitlig halvautomatisk analys av encelliga krafter - med tanke på att ett typiskt experiment kan kräv...

Mekano känsliga cellbaserade analyser tillåter för att undersöka vidhäftande celler, med ett brett spektrum av tillämpningar som återspeglar den centrala roll som mekaniker kan spela i cellbiologi. Dessa program fokuserar ofta på de bakomliggande mekanismer som driver subcellulära processer eller hela-cellens beteende. Å ena sidan, kan yttre miljöfaktorer såsom extracellulär matriskomposition eller matrisstelhet dramatiskt påverka den mekaniska och biologiska responsen av en cell. 1 Detsamma kan observeras efter användning av många klasser av farmaceutiskt aktiva föreningar, vars effekter är kännetecknas ofta med hjälp av cellodlingsmodeller. 2 Å andra sidan genotypiska egenskaper, såsom de som orsakas av spontana eller experimentellt inducerad genetiska mutationer, kan inducera markerade förändringar i cellfenotyp som är associerade med förändringar i cytoskelettet struktur och funktion. 3 Dessa exempel är bara några av de många möjligaämnen för vilka mekanisk fenotypning av celler är relevant, och alla dessa har fördel undersökts med micropost matriser. Vid tidpunkten för denna skrift, har cirka 200 artiklar publicerats som beskriver cell micropost interaktion. Dessa arbeten diskutera teoretiska aspekter av micropost böjnings principer samt praktiska anvisningar om deras tillverkning. Den första artikeln beskriver samspelet mellan celler och flexibla micropost matriser publicerades av Tan och kollegor under 2003. 4 I motsats till klassisk dragkraft mikroskopi (TFM) där kontinuerliga mjuka underlag används för att uppskatta nanonewton skala cellkontraktilitet, Tan et al. beskrivit ett förfarande med hjälp av flera tätt åtskilda vertikala balkar av silikonelastomer. De främsta fördelarna med denna teknik dyker upp från två viktiga funktioner. Först för att ändra cell uppenbara substrat styvhet behöver man bara ändra micropost dimensioner samtidigtsubstratkompositionen annars konstant och därmed undvika skillnader i ytan topologi och kemi. Andra microposts agera som enskilda fjädrar som kan diskret analyseras med kraft och rumsliga resolutioner om i storleksordningen enskilda fokala sammanväxningar och kan minska de analytiska utmaningar som är förbundna med liknande analys av standard TFM. Idag utbudet av applikationer för micropost arrayer vida överstiger bara kartläggning av krafterna för några enstaka celler. Exempelvis Akiyama rapporterar användningen av en isolerad dorsala kärl vävnad från en mal caterpillar som ett manövreringsorgan för en micropost matris, i syfte att utveckla en insekt muskeldriven autonom mikro roboten. 5 Emellertid har de flesta publicerade tillämpningar av microposts fokuserat på studier av medicinska tillstånd som infektion eller cancer. Exempelvis har micropost arrayer använts för att studera kraftgenereringen av medföljande typ IV pili av Neisseria gonorrhOEA kolonier som är associerad med signalkaskader öka infektion. 6 Andra har använt microposts att studera celler bröstcancer som behandlats med läkemedelssubstanser som riktar cytoskelettet. 7 Avböjning av en micropost beskrivs ofta med användning av klassisk balkteori för en konsol med en ändbelastning med antagande cellen fäster endast till den yttersta spetsen av den micropost. Här den anbringade kraften F som orsakar en nedböjning δ beror på micropost s &quot;böjstyvhet&quot; k och beräknas genom: <img alt="Ekvation 1" src="/files/ftp_upload/53350/53350eq1.jpg" /> (1) med E, I och L är Youngs modul, område tröghetsmoment och bomlängd respektive. Men resultaten från denna ekvation ger endast en allmän uppskattning av de krafter i arbetet, eftersom strålen klippning och bockning samt substrat skevhet inte tas i acräkna. Med tanke på att microposts är oftast gjorda av mjuka material som polydimetylsiloxan (PDMS) -baserade silikongummi dessa faktorer måste tas med. . Schoen m.fl. visade att det finns en sådan korrektionsfaktor baserad på bildformat för micropost (L / D) och motsvarande polymerens Poisson förhållande v 8 Det ges av.: <img alt="Ekvation 2" src="/files/ftp_upload/53350/53350eq2.jpg" /> (2) Med T tilt (v) är en tiltkoefficient som innehåller passande parameter a = 1.3 som kan hittas i samma artikel: <img alt="Ekvation 3" src="/files/ftp_upload/53350/53350eq3.jpg" /> (3) Det innebär en micropost rättade styvhet k corr är produkten av den rena böj styvhet k = k böj och korrektionsfaktorn corrgetts av: <img alt="Ekvation 4" src="/files/ftp_upload/53350/53350eq4.jpg" /> (4) Därför bör cellkraftberäkningar utföras med mer förfinad variant av ekvation (1) nu läser: <img alt="Ekvation 5" src="/files/ftp_upload/53350/53350eq5.jpg" /> (5) Effekterna av korrigeringen blir mer uppenbara så snart som typiska värden för micropost dimensioner används. Till exempel, en 15-micron långa micropost med ett cirkulärt tvärsnitt och en diameter av 5 pm gjord av PDMS-baserade silikongummi leder till en korrektionsfaktor på 0,77 och därmed ett okorrigerat beräkning skulle överskatta utövade cellkrafterna med 23%. Detta blir ännu allvarligare för microposts med mindre bildformat. Traditionellt har micropost bildanalys även baserats på idealiserade balkböjning teori. År 2005 den grupp som börjat använda sig av MICRopost arrayer publicerade en bildanalysprogram lämpad för micropost analys 9 Programmet kräver en programvarulicens och användaren måste ta tre bilder för varje position. en vardera från micropost s topp- och bottenplan i sändningsläge och en annan i fluorescensläge med den färgade cellen. Efter att ha jämfört de övre och nedre positionerna för varje micropost mjukvaran bestämmer en kraftvektorfält och beräknar relaterade parametrar såsom kraft per tjänst. Andra mjukvarupaket finns och deras analys principer kortfattat nämns i motsvarande artiklar som beskriver dem, men dessa analytiska programvarupaket är i allmänhet inte tillgängliga för allmänheten. 10,11 De micropost matriser avsedda för kartläggning cell krafter kan klassificeras som antingen är i ett ortogonalt micropost layout eller en hexagonal en, den senare som har fördelen av ekvidistanta mellanrum mellan alla grann microposts. Typiska microposts hektarve ett cirkulärt tvärsnitt och deras dimensioner varierar från 1,0 | im till 10 | j, m i diameter och två till 50 ^ m i längd. 4 emellertid microposts med elliptiskt eller kvadratiskt tvärsnitt har också rapporterats. 12,13 Användningen av PDMS-baserade silikonblandningar som micropost material medger att lägga till nanopartiklar i blandningen. Till exempel att lägga till kobolt nanostavar möjliggör en magnetisk aktivering av micropost och därmed ger ett annat frihetsgrad till potentiella experimentell design. 14 De flesta grupper producera sina micropost arrayer på plana styva substrat såsom skyddsglas eller inuti en petriskål. Men Mann och medarbetare rapporterade nyligen en micropost array bildas på ett töjbart membran. 15 Detta gör att tillämpningen av cellsträckkrafter till vidhäftande celler medan de studerar levande celler subcellulär dynamiska svar när det gäller cellkontraktilitet. Den allmänt använda och mest om inrättandeinrättats förfarande för framställning micropost matriser är baserad på mjuk litografi, såsom beskrivs i de insikts protokollen för Sniadecki och kollegor. 16-18 I korta standardrenrumsprocesser används för att generera mikrostrukturerna på toppen av en kiselskiva med användning SU8 fotoresist. Detta följs av en kopieringsprocess, vari silikongummit gjuts över de strukturer överföra dem i formar. I ett andra steg är dessa formar användes för att replikera den initiala mikrostruktur med användning av silikongummi på toppen av ett valt substrat. Men trots det stora och växande antal publikationer relaterade till deras ansökan om upprättande av ett tillverkningsprocess för microposts tar avsevärd tid även för mikrotekniska experter, det finns många processteg som kräver optimering och anpassning till den specifika labbmiljö och micropost layout för att ge en acceptabel kvalitetsnivå. Kommersiella micropost arrayer finns nu i en färdig-to-användning (&quot;off-the-shelf&quot;) format med en genomgående hög kvalitet. Som sådana är de ett alternativ till de komplexa och tidskrävande tillverkningsprocess som krävs för tillverkning på plats. I denna uppsats en kommersiellt tillgänglig micropost array användes för att kartlägga cellulära krafter med hjälp av en enda ljusa fält mikroskopi bild. Ännu viktigare här artikeln beskriver och dokumenterar en fullt fungerande öppen källkod som heter MechProfiler, som är tillgänglig för nedladdning som kompletterande material till detta manuskript. En aktivt underhållna version av programvaran kan också hittas på http://www.orthobiomech.ethz.ch. Kombinationen av en &quot;off-the-shelf&quot; analys och en kompatibel öppen källkod analysprogram sänker markant posten hindret för att uppnå korrekta TFM experiment. Forskare utan tillgång till vare sig renrumsfaciliteter eller mjukvaruutveckling expertis kan analysera cellulära krafter framgångsrikt. Det gör det möjligt för en användare att inrikta sig på de mechanosensitivity analys utgång snarare än tekniken i sig, och gör dragkraft mätningar tillgängliga för en bredare gemenskap. Dessutom är detta ett viktigt steg för att bana väg för helautomatisk screening av micropost matriser. Den MechProfiler analysprogram behandlar bilder i filformatet tiff, png, bmp och jpg. Bilderna kan tas med användning av fluorescens, faskontrast eller ljusfält Ijusmikroskopi. Den fristående program körs tillsammans med den fria Matlab Compiler Runtime (tillgänglig på: Figur 12) och underliggande algoritmer möjliggör strömlinjeformad bildbehandling, vilket gör det möjligt för användaren att bearbeta bilder med enkla eller multipla celler i ca 1 min. Vidare kan dessa celler antingen levande eller &quot;fasta&quot;. Den MechProfiler programvara kan kraftigt öka dataanalys genomströmningen genom att förlita sig på reproducerbarhet kvalitet kommersiella micropost arrayer, närmare bestämt standard &amp; #8220, icke-böjs &quot;position varje inlägg i gruppen kan antas mot en idealisk galler (avvikelser för nätet i grupperna som används i denna studie var mindre än 100 nm). Kort sagt en öppnar ett urval av bildfiler för analys, beskär dem till regionen av intresse, definierar de tjänster som omfattas av celler eller som måste kasseras, bestämmer postpositioner, beräknar deflektioner / krafter mot den ideala nätet, och slutligen sparar alla cellspecifika data med möjlighet till export, bland annat till en vanlig kontors kalkylblad.

<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.

Den största fördelen med den beskrivna tekniken ligger i dess enkelhet och potential för snabb och effektiv integrering i den rutinmässiga laborationer. Kombinationen av högkvalitativa kommersiella sensorsystem parat med öppen källkod ger information om mekano-känslighet som annars skulle kräver tillgång till renrumsfaciliteter och fördjupad kunskap om bildanalys och mjukvaruutveckling. Figur 1 visar arbetsflödet för den presenterade metoden. Det börjar med att förbereda och sådd celler ...

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.

Enkelt och exakt Mechano-profilering på 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.

Studera Cell Rolling Trajectories på asymmetrisk Receptor Mönster

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).

Hög effektivitet, Platsspecifik transfektion av vidhäftande celler med siRNA Använda mikroelektrod 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.

Förslutningsbar Femtoliter Chamber arrayer för Cellfri biologi

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-kluster arrayer i en stöds lipiddubbelskikt

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.

Gränssnitt mikrofluidik med mikroelektrod matriser för att studera Neuronal kommunikation och Axonal signalen förökning

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.

Humana iPSC-härledda Kardiomyocyte nätverk på Multiwell Micro-elektrod arrayer för återkommande åtgärder potentiella inspelningar

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.

Tillverkning av Ti3C2 MXene Microelectrode Arrays för 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.

Substructure Analyzer: Ett användarvänligt arbetsflöde för snabb utforskning och korrekt analys av cellulära organ i fluorescensmikroskopi bilder

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.

Högdimensionell flödescytometri för immunfunktionsanalys av dissekerade implantatvävnader

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.

Tillverkning av 3D Cardiac Microtissue Arrays med mänskliga iPSC-härledda kardiomyocyter, hjärtfibblaster och endotelceller

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