Diese Arbeit wurde vom Institut Curie, Paris, Frankreich gegründet. Die mechanische Bahre wurde von Damien Cuvelier (Institut Curie) entworfen und wird von GREM hergestellt (mecanique-grem.com). Die Strukturierung auf PDMS wurde von Ammar Azioune (Bordeaux II University) entwickelt.

1. Passivierung der PDMS <ol><li> Ein Stück des PDMS ca. 35 mm x 20 mm aus einer bereits geformten Folie (zum Beispiel GelPak, wie in der Tabelle aufgeführten Materialien). </li><li> Entfernen Sie die obere und die untere Schutzschichten aus Kunststoff (falls erforderlich) und mit Hilfe einer Pinzette, um die PDMS in einer Kunststoff legen (nicht zellkulturbehandelte) Petrischale. </li><li> Waschen Sie die PDMS mit 70% Ethanol für 5 Minuten auf einem Schüttler bei 30 Schwingungen / min. </li><li> Trocknen Sie die Oberfläche durch strömende Luft auf sie. </li><li> Illuminate mit tiefen UV (λ = 180 nm) für 5 min bei einem Abstand von den UV-Lampen von etwa 5 cm (siehe Materialien Blatt für Lampe Referenz; Parameter für verschiedene Lampen variieren). </li><li> Planen 200 ul EDC / NHS-Lösung für jedes Stück PDMS (muss unmittelbar vor der Verwendung hergestellt, da die Reaktivität der Lösung zerfällt in wenigen Stunden werden). Für 1 ml 0,05 M MES + 0,5 M NaCl-Puffer bei pH 6,0, fügen 11,5 mg Sulfo-NHS und 19,2 mg EDC. </li><li> Übertragen Sie diePDMS Blätter von der Petrischale mit dem Deckel der Petrischale, die nicht belichtet worden ist. Dadurch wird sichergestellt, dass die Umgebung des PDMS sind sehr hydrophob und ermöglichen den nächsten Schritt. </li><li> In EDC / NHS-Lösung und Inkubation für 15 min bei Raumtemperatur. </li><li> Spülen EDC / NHS mit Wasser. </li><li> Hinzufügen PLL-g-PEG-Lösung (0,5 mg / ml in 10 mM HEPES pH 8.6) und Inkubation von 3 h bis über Nacht bei Raumtemperatur. </li><li> Spülen Sie die PLL-g-PEG mit Wasser. Passi PDMS (mit PLL-g-PEG funktionalisiert) kann für mehrere Tage bei 4 ° C gelagert werden </li></ol> 2. Strukturieren der PDMS <ol><li> Nehmen Sie ein PDMS Blatt und legen Sie es auf einem synthetischen Quarzphotomaske tragen die Mikroformen zur Strukturierung (siehe Abbildung 1). Legen Sie die PDMS seitigen Lager die PLL-g-PEG mit Blick auf den Chrom Seite der Photomaske. </li><li> Beleuchten 7 Minuten durch die Photomaske in einem Abstand von der UV-Lampen von etwa 5 cm.</li><li> Fügen Sie Wasser auf die Maske + PDMS und schälen Sie die PDMS langsam aus der Maske. </li><li> Inkubieren mit Fibronectin-Lösung bei 50 mg / ml in HEPES-Puffer bei pH 8,6 für 1 Stunde bei Raumtemperatur. Es ist möglich, andere ECM-Proteine ​​zu verwenden, aber diese sind nicht mit diesem Protokoll getestet. </li><li> Spülen mit PBS. </li></ol> 3. Das Gerät montieren <ol><li> Montieren Sie die zuvor passivierten PDMS auf die Spannvorrichtung (siehe Diskussion zur Fehlersuche). <ol><li> Befestigen einer Seite des PDMS zu dem feststehenden Teil der Trage. </li><li> Befestigen Sie die andere Seite mit dem Mobilteil der Trage ohne Spann zu viel von der PDMS Blatt. </li></ol></li><li> Schneiden Sie ein Rechteck von PDMS (22 mm x 19 mm) in einer dicken PDMS Stich-und Schnitt ein weiteres Rechteck im Inneren, um einen Pool (oder Rahmen) haben, die die Zellen-und Medien behalten werden (siehe Abbildung 2). </li><li> In Silikonfett unter diese PDMS Pool und legen Sie es auf der PDMS-Blatt zu erstellen med.ium Haltebecken. Das Fett wird die Gleitfähigkeit des Pools über das PDMS Blatt beim Strecken zu ermöglichen. </li></ol> 4. Strukturieren der Zellen <ol><li> Lösen Sie die Zellen aus einer 50% Konfluenz Kulturflasche mit Versene. </li><li> Zähle die Zellen zu resuspendieren und diese in einer Konzentration von 200.000 Zellen / ml. </li><li> 1 ml der Zellsuspension in den Pool (siehe Diskussion für weitere Details). </li><li> Sei (je nach Zelltyp, RPE-1-Zellen werden 10 min und HeLa-Zellen 20 min dauern) die Zellen zu binden, um die Muster für 10-30 min. </li><li> Vorsichtig spülen Sie die schwimmenden Zellen mit ins Gleichgewicht Medium (ins Gleichgewicht mittel Inkubator). </li><li> Lassen Sie die Zellen verteilt für ein paar Stunden auf die Muster (RPE-1 ist mindestens 2 Stunden brauchen, HeLa-Zellen wird 3 Stunden brauchen). </li><li> Fügen Sie ein Deckglas auf den Pool, um Verdunstung und mittlere Leckage bei der PDMS Pausen zu vermeiden. </li><li> Legen Sie das Gerät auf einem inversen Mikroskop und starten Bildgebung. Vermeiden Sie die Verwendung von Ölimmersion objectives, da es nicht durch Fokussierung Probleme funktionieren. </li></ol> 5. Dehnung <ol><li> Die Mikrometerschraube drehen, während die Korrektur der Tischposition in x-, y-und z-Achse (hauptsächlich x). Die Bühnenposition muss korrigiert, die Erweiterung des PDMS und den Verlust des Fokus entgegenzuwirken. (Siehe &quot;Bei der Strecke&quot; Absatz in der Diskussion). </li></ol>

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

Die Autoren erklären, keine finanziellen Interessen konkurrieren.

Obwohl diese Technik wurde mehrfach verwendet und wird gründlich getestet, gibt es mehrere kritische Schritte, die zu einem fehlgeschlagenen Experiment führen kann. Über die PDMS: Für diese Arbeit GelPak ein handelsüblicher Dünn PDMS Blatt, verwendet. Alternativ PDMS Blätter können direkt von PDMS-Mischung gegossen werden. Wir empfehlen die Verwendung GelPak, weil es mehr reproduzierbar, und ist weniger wahrscheinlich zu brechen Vergleich zu maßgeschneidert...

Die Zellen Zusammensetzen eines Gewebe in höheren Organismen unterliegen mechanischen Spannungen und Dehnungskräfte kommen entweder von der äußeren Umgebung oder von umgebenden Zellen 1,2. Die Zellen müssen sich anzupassen und zu widerstehen, diese Kräfte, um die Gewebeintegrität aufrecht zu erhalten. Solche Kräfte sind auch für Gewebe-Morphogenese während der Entwicklung 3,4. Anwenden von mechanischen Kräften auf kultivierte Zellen ist ein Weg, zu imitieren, was in einem Gewebe passieren könnte, aber mit einer quantitativen und unabhängige Steuerung der Zellform und Zellverformung 5,6. Hierzu könnten verschiedene Techniken verwendet werden. Man kann auf die Zellen (die ganze Zelle oder einen Teil davon), beispielsweise mit AFM oder Derivate 7,8 oder strecken das Substrat die Zellen wachsen auf zu drücken. Die in diesem Dokument beschriebenen Verfahren zeigt, wie ein Flugzeug Substrat mit Zellen plattiert dehnen. Auf m Diese Technik wurde ursprünglich entwickelt, um beurteilen die Rolle der Streitkräfte ausgeübtitotic Säugetierzellen 9. Mitotische Zellen bleiben mit dem Substrat verbunden, durch Zurückziehen und Strecken der Fasern Membran ausgeübt wird, eine Kraft auf jene Fasern, die wiederum provozierte die Rotation der mitotischen Spindel. Das Interesse der Kombination von Klebstoff und Mikro Recken um eine unabhängige Steuerung von Kräften und Form der einzelnen Zellen zu erreichen. Es ist beispielsweise möglich, eine eiförmige Zelle in eine perfekt runde Form isotropen strecken, während einachsige Dehnung angewendet wird. Wenn die Zellen nicht auf Mikro geflochten, uniaxiales Strecken Ergebnisse in Zellstreckung, wobei die meisten Zellen, die ein mit dem Streckachse ausgerichtet langen Achse. Es ist dann schwierig, den Effekt der Ausrichtung der langen Achse und der Wirkung der Ausdehnung auf die Zellen zu trennen. Das Gerät eignet sich für alle lebenden Zellen, einschließlich Langzeitablauf Fluoreszenz-Mikroskopie, und Medikamente können während des Experiments hinzugefügt werden. Die tiefe Mikrostrukturierungsverfahren 10 UVswurde im Detail in Azioune et al. 11 Musterung auf PDMS wurde beschrieben in Azioune et al. 12 Die vorliegende Streck Protokoll ist ein Video-Version Carpi et al. 13

<table><tbody><tr><td>GelPak</td><td>GelPak</td><td>PF-60-X4</td><td>Different thickness/stickiness are available. One alternative could be to cast your PDMS&amp;nbsp;yourself.</td></tr><tr><td>Silicon grease</td><td>GE Bayer Silicones</td><td>Baysilone-Paste</td><td>This one is biocompatible</td></tr><tr><td>Stretching device</td><td>GREM m&amp;eacute;canique</td><td>Stretcher 2011</td><td>&amp;nbsp;</td></tr><tr><td>EDC (N-(3-Dimethylaminopropyl)-N&amp;prime;-ethylcarbodiimide hydrochloride)</td><td>Sigma</td><td>3450</td><td>Stable 6 months at -20 &amp;deg;C</td></tr><tr><td>NHS (N-Hydroxysulfosuccinimide sodium salt)</td><td>Sigma</td><td>56485</td><td>Protect from humidity</td></tr><tr><td>Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td><td>SurfaceSolutions (Zurich)</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Synthetic Quartz photomask</td><td>Toppan</td><td></td><td>Take standard binary photomask in Quartz</td></tr><tr><td>Fibronectin from bovine plasma</td><td>Sigma</td><td>F1141</td><td>&amp;nbsp;</td></tr></tbody></table>

stretching micropatterned cells on a pdms membrane

Mechanische Kräfte auf Zellen ausgeübt und / oder Gewebe spielen eine wichtige Rolle in zahlreichen Prozessen. Wir haben ein Gerät an Zellen auf einer Polydimethylsiloxan (PDMS)-Membran, mit bildgebenden kompatibel vernickelt strecken entwickelt. Dieses Verfahren ist reproduzierbar und vielseitig. Die PDMS-Membran, um Zellen oder Gewebe zu einem bestimmten Geometrie beschränken mikrostrukturiert werden. Der erste Schritt ist die Mikrostrukturen auf den PDMS-Membran mit einem tiefen UV-Technik gedruckt. Die PDMS-Membran wird dann auf einer mechanischen Trage befestigt. Eine Kammer ist auf der Oberseite der Membran mit biokompatiblen Fett damit Gleiten während der Strecke gebunden. Die Zellen werden ausgesät und für mehrere Stunden auf den Mikro verbreiten. Die Probe kann mehrere Male unter Verwendung einer Mikrometerschraube gespannt und ungestreckte werden. Es dauert weniger als eine Minute, um die Strecke in vollem Umfang (rund 30%) anzuwenden. Die hier vorgestellte Technik nicht eine motorisierte Vorrichtung, die für eine erforderliche umfassenpplying wiederholter Dehnungszyklen schnell und / oder Computer-kontrolliertes Recken, aber dies kann realisiert werden. Dehnen von Zellen oder Gewebe kann von Interesse für die Fragen, um die Zellkräfte, Zell-Antwort gegen mechanische Beanspruchung oder Gewebemorphogenese bezogen werden. Diese Video-Präsentation wird zeigen, wie die typischen Probleme, die, wenn Sie diese Art von scheinbar einfachen Experiment entstehen könnten.

<html> Die in diesem Video-Protokoll vorgestellte Technik erlaubt die Anwendung von Kräften auf die Rückzugs Fasern mitotischer Säugerzellen. In der Tat, während der Zellteilung, bei der mitotischen Phase, Säugetierzellen zurückziehen, um die Form einer Kugel nehmen und hinterlassen dünnen Aktin-Kabel von Membran umgeben, die auf dem Substrat befestigt sind. Diese Kabel (Rückzug Fasern), sind die Erinnerung an die Zellgeometrie, bevor er in Division. Machen Mikro mit tiefen UV durch eine Photomaske auf PDMS Dünn...

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

Stretching Micropatterned Cells on a PDMS Membrane

Dieses Manuskript stellt eine Technik, um auf adhärenten Zellen oder Gewebe gelten oder freiKräfte mit unidirektionalen Stretching.

Stretching-Cells auf einer mikrostrukturierten PDMS-Membran

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

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

Research

JoVE Journal

Bioengineering

15.2K Aufrufe.  Institut Curie.  Dieses Manuskript stellt eine Technik, um auf adhärenten Zellen oder Gewebe gelten oder freiKräfte mit unidirektionalen Stretching. 

Serie: Stretching Micropatterned Cells on a PDMS Membrane

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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

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

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

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

Forschung

Biotechnik

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

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

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

Stencil Mikrostrukturierung von humanen pluripotenten Stammzellen für die Sondierung räumliche Organisation Differenzierung Fates

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

Entwicklungsbiologie

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

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

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

A Novel Stretching-Plattform für Anwendungen in der Zell-und Gewebe Mechanobiologie

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

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

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

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

Strukturierung von Mikroorganismen und Mikropartikeln durch sequentielle kapillaritätsunterstützte Montage

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

Ingenieurwesen

A Versatile Method of Patterning Proteins and Cells

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

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

Eine vielseitige Methode von Patterning Proteinen und Zellen

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

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Einfache, erschwingliche und modulare Musterung von Zellen mit DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Pattern Generation for Micropattern Traction Microscopy

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

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

Mustergenerierung für die Mikromuster-Traktionsmikroskopie

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

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

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

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

Erzeugung von Multicue-Zellmikroumgebungen durch UV-Photostrukturierung von dreidimensionalen Zellkultursubstraten

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Eine neuartige Methode zur strukturierung von tinten- und markierungsfreien magnetischen Archimedes-Zellen

Cell Patterning Using Magnetic-Archimedes Strategy

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell patterning strategy based on the Magnetic-Archimedes (Mag-Arch) effect is introduced. This approach enables precise control of cell distribution without the use of ink materials or labeling particles. By introducing a paramagnetic reagent to enhance the magnetic susceptibility of the cell culture medium, cells are repelled by magnets and arrange themselves into a pattern complementary to the magnet sets positioned beneath the microfluidic substrate.
In this article, detailed procedures for cell patterning using the Mag-Arch-based strategy are provided. Methods for patterning single-cell types as well as multiple cell types for co-culture experiments are offered. Additionally, comprehensive instructions for fabricating microfluidic devices containing channels for cell patterning are provided. Achieving this feature using parallel methods is challenging but can be done in a simplified and cost-effective manner. Employing Mag-Arch-based cell patterning equips researchers with a powerful tool for in vitro research.

Autor im Rampenlicht: Magnetbasierte Zellstrukturierungsmethode für biomedizinische Hochdurchsatzanwendungen 

This protocol describes an ink-free, label-free, substrate-independent, high-throughput cell patterning method based on the Magnetic Archimedes effect.