Name: Stretching Micropatterned Cells on a PDMS Membrane (Video) | JoVE Journal
Uploaded: 2014-01-22T13:45:00.000+00:00
Duration: 9 min 41 s
Description: Learn to stretch micropatterned cells on a PDMS membrane for studying cellular responses to mechanical forces. This technique enables controlled force application on adherent cells using video microscopy.

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5 minutes

This work was founded by Institut Curie, Paris, France. The mechanical stretcher was designed by Damien Cuvelier (Institut Curie) and is manufactured by GREM (mecanique-grem.com). The patterning on PDMS was developed by Ammar Azioune (Bordeaux II University).

1. Passivation of the PDMS
<ol>
	<li>Cut a piece of PDMS approximately 35 mm x 20 mm from a pre&#160;made sheet (for instance, GelPak, as listed in the table of materials).</li>
	<li>Remove the top and the bottom protective layers of plastic (if necessary) and use tweezers to place the PDMS in a plastic (not cell culture treated) Petri dish.</li>
	<li>Wash the PDMS with 70% ethanol for 5 min on a rotator at 30 oscillations/min.</li>
	<li>Dry the surface by flowing air on it.</li>
	<li>Illuminate with deep UV (&#955; = 180 nm) for 5 min at a distance from the UV bulbs of about 5 cm (see Materials sheet for lamp reference; parameters will vary for different lamps).</li>
	<li>Prepare 200 &#181;l&#160;of EDC/NHS solution for each PDMS piece (this must be prepared just before use because the reactivity of the solution decays in a matter of hr). For 1 ml of 0.05 M MES + 0.5 M NaCl buffer at pH 6.0, add 11.5 mg of Sulfo-NHS and 19.2 mg of EDC.</li>
	<li>Transfer the PDMS sheets from the Petri dish to the lid of the Petri dish, which has not been illuminated. This will ensure that the surroundings of the PDMS are very hydrophobic and facilitate the next step.</li>
	<li>Add EDC/NHS solution and incubate for 15 min at room temperature.</li>
	<li>Rinse EDC/NHS with water.</li>
	<li>Add PLL-g-PEG solution (0.5 mg/ml in HEPES 10 mM pH 8.6) and incubate from 3 hr to overnight at room temperature.</li>
	<li>Rinse the PLL-g-PEG with water. Passivated PDMS (functionalized with PLL-g-PEG) can be stored for several days at 4&#160;&#176;C.</li>
</ol>
2. Patterning of the PDMS
<ol>
	<li>Take a PDMS sheet and place it on a synthetic quartz photomask bearing the microfeatures for patterning (see Figure 1). Place the PDMS side bearing the PLL-g-PEG facing the chrome side of the photomask.</li>
	<li>Illuminate for 7 min through the photomask at a distance from the UV bulbs of about 5 cm.</li>
	<li>Add water onto the mask + PDMS and peel the PDMS slowly off the mask.</li>
	<li>Incubate with fibronectin solution at 50 mg/ml in HEPES buffer at pH 8.6 for 1 hr at room temperature. It is possible to use others ECM proteins, but these have not been tested with this protocol.</li>
	<li>Rinse with PBS.</li>
</ol>
3. Mounting the Device
<ol>
	<li>Mount the previously passivated PDMS onto the stretching device (see Discussion for troubleshooting).
	<ol>
		<li>Attach one side of the PDMS to the fixed part of the stretcher.</li>
		<li>Fix the other side to the mobile part of the stretcher without clamping too much of the PDMS sheet.</li>
	</ol>
	</li>
	<li>Cut a rectangle of PDMS (22 mm x 19 mm) in a thick PDMS stab and cut another rectangle inside in order to have a pool (or frame) that will retain the cells and media (see Figure 2).</li>
	<li>Add silicone grease under this PDMS pool and place it on top of the PDMS sheet to create a medium&#160;retaining pool. The grease will allow the gliding of the pool over the PDMS sheet during stretching.</li>
</ol>
4. Patterning the Cells
<ol>
	<li>Detach the cells from a 50% confluency culture flask with Versene.</li>
	<li>Count the cells and resuspend them at a concentration of 200,000 cells/ml.</li>
	<li>Add 1 ml of cell suspension in the pool (see Discussion for additional details).</li>
	<li>Let the cells bind to the patterns for 10-30 min (depending on the cell type, RPE-1 cells will take 10 min and HeLa cells 20 min).</li>
	<li>Gently flush the floating cells with equilibrated medium (equilibrate medium in incubator).</li>
	<li>Let the cells spread for a few hr on the patterns (RPE-1 will need at least 2 hr; HeLa cells will need 3 hr).</li>
	<li>Add a coverslip on top of the pool to avoid evaporation and medium leakage in case the PDMS breaks.</li>
	<li>Put the device on an inverted microscope and start imaging. Avoid the use of oil immersion objectives as it will not work due to focusing problems.</li>
</ol>
5. Stretching
<ol>
	<li>Turn the micrometric screw while correcting the stage position in the x-, y-,&#160;and z-axis (mainly x). The stage position needs to be corrected to counteract the widening of the PDMS and the loss of focus. (See &#34;During the stretch&#34; paragraph in the DISCUSSION).</li>
</ol>

<ol><li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+force+and+geometry+sensing+regulate+cell+functions%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">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/pubmed?term=((Mechanics+of+epithelial+tissue+homeostasis+and+morphogenesis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Mechanical+control+of+morphogenesis+by+Fat%2FDachsous%2FFour-jointed+planar+cell+polarity+pathway%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+influence+of+cell+mechanics%2C+cell-cell+interactions%2C+and+proliferation+on+epithelial+packing%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">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/pubmed?term=((Nature+and+anisotropy+of+cortical+forces+orienting+Drosophila+tissue+morphogenesis%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Real-time+single-cell+response+to+stiffness%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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/pubmed?term=((Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((External+forces+control+mitotic+spindle+positioning%5BTitle%5D)+AND+%22Nat.+Cell.+Biol%22%5BJournal%5D)">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li>
<li>Azioune, A., Storch, M., Bornens, M., Théry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+and+rapid+process+for+single+cell+micro-patterning%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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., Théry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+micropatterns%3A+A+direct+printing+protocol+using+deep+Uvs%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Micropatterning+on+silicon+elastomer+%28PDMS%29+with+deep+UVs%5BTitle%5D)+AND+%22Protoc.+Exch%22%5BJournal%5D)">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/pubmed?term=((Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li>
</ol>

The authors declare no competing financial interests.

Although this technique has been used numerous times and is thoroughly tested, there are several critical steps that can lead to a failed experiment.
About the PDMS:
For this work, GelPak, a commercially available thin PDMS sheet, was used. Alternatively PDMS sheets can be cast directly from PDMS mix. We recommend using GelPak because it is more reproducible, and is less likely to break compared to custom made PDMS.
About the device:
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The cells composing a tissue in higher organisms are subject to mechanical tensions and stretching forces coming either from the external environment or from surrounding cells1,2. Cells must adapt to and resist these forces in order to maintain tissue integrity. Such forces are also important for tissues morphogenesis during development3,4. Applying mechanical forces on cultured cells is a way to mimic what might happen in a tissue, but with a quantitative and independent control of cell shape and cell deformation5,6. For this, several techniques might be used. One can press on the cells (the whole cell or part of it), for example using AFM or derivatives7,8&#160;or stretch the substrate the cells are growing on.
The method described in this paper demonstrates how to stretch a plane substrate plated with cells. This technique was originally developed to assess the role of forces exerted on mitotic mammalian cells9. Mitotic cells stay connected to the substrate through retraction fibers and stretching the membrane exerted a force on those fibers, which in turn provoked the rotation of the mitotic spindle. The interest of combining adhesive micropatterns and stretching is to achieve independent control of forces and shape of individual cells. It is for example possible to stretch an ovoid cell into a perfectly isotropic round shape, while uniaxial stretch is applied. If the cells are not platted on micropatterns, uniaxial stretching results in cell elongation, with most cells having a long axis aligned with the stretch axis. It is then difficult to separate the effect of the long axis alignment and the effect of the stretch applied to the cells.
The device is suitable for any live cell imaging, including long time lapse fluorescent microscopy, and drugs can be added during the experiment. The deep UVs&#160;micropatterning method10&#160;was described in details in Azioune et al.11&#160;Patterning on PDMS was described in Azioune et al.12&#160;The present stretching protocol is a video version of 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

Stretching Micropatterned Cells on a PDMS Membrane

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 PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

The technique presented in this video protocol allowed the application of forces on the retraction fibers of mitotic mammalian cells. Indeed, during cell division, at the mitotic stage, mammalian cells retract to take the shape of a sphere and leave behind thin actin cables surrounded by membrane which are attached to the substrate. These cables (retraction fibers), are the memory of the cell geometry before going into division. Making micropatterns with deep UV through a photomask on PDMS thin film (Figure 1</st...

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

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 ...

The overall goal of this procedure is to stretch pattern cells on an elastic membrane of poly dimethyl suboxane or PDMS. This is accomplished through the ation of A-P-D-M-S sheet, followed by the application of a micro pattern on the PDMS. The micro patterned PDMS sheet is then mounted on the stretching device and a medium retaining pool is placed on top.Next, the cells are plated on the surface of the micro patterned PDMS sheet and allowed to attach to the pattern before flushing to remove unattached cells. The final step is to apply an elongation to the cell containing PDMS to stretch the cells. Ultimately, video microscopy is used to show that application of forces on the retraction fibers of mitotic mammalian cells in cell stretching results in deformation of the cell.This method can help answer key questions in the biomechanics field, such as what are the effects of external forces on cellular processes like division? We first had the idea for this method when we wanted to apply forces on a dividing cell to begin cut a piece of PDMS approximately 35 by 20 millimeters from a pre-made sheet. Here, a commercially available thin PDMS sheet is used because it is more reproducible and is less likely to break as compared to custom-made.PDMS.Remove the top and the bottom protective layers of plastic and use tweezers to place the PDMS in a plastic petri dish. Then wash the PDMS with 70%ethanol for five minutes on a rotator at 30 oscillations per minute. Following the wash, dry the surface by flowing air over it.Next illuminate the PDMS with deep ultraviolet light for five minutes at a distance from the ultraviolet bulbs of about five centimeters. Meanwhile, prepare an E-D-C-N-H-S solution as described in the text protocol. The solution must be prepared just before use because the reactivity of the solution decays in a matter of hours.Transfer the PDMS sheets from the Petri dish to the lid of the Petri dish, which has not been illuminated. This will ensure that the surroundings of the PDMS are very hydrophobic and facilitate. The next step.Pipette the E-D-C-N-H-S solution over the PDMS and incubate for 15 minutes at room temperature. Following the incubation. Rinse the excess E-D-C-N-H-S off with water.Next, add the PLL grafted PEG solution and incubate from three hours to overnight at room temperature following incubation, rinse the excess PLL grafted peg off the PDMS with water. The PDMS is now passivated or functionalized with PLL grafted PEG and can be stored for several days at four degrees Celsius. To pattern the PDMS place, A-P-D-M-S sheet on a synthetic quartz photo mask bearing the micro features for patterning.Place the PDMS side bearing the PLL grafted PEG facing the chrome side of the photo mask. Then illuminate for seven minutes through the photo mask at a distance from the ultraviolet bulbs of about five centimeters. Following illumination.Add water onto the mask and PDMS then peel the PDMS slowly off the mask mount the previously passivated PDMS onto the stretching device. Attach one side of the PDMS to the fixed part of the stretcher. Fix the other side to the mobile part of the stretcher without clamping too much of the PDMS sheet.If the PDMS is clamped too much, it becomes shorter and this can lead to braking as the tension will be greater for the same distance of stretch. Make sure the screws are well tightened, or the PDMS sheet will slip as soon as the stretch begins, or later on during the experiment. Next, incubate the PDMS with fibronectin solution for one hour.At room temperature, it is possible to use other extracellular matrix proteins, but these have not been tested with this protocol. As a final step, rinse the PDMS with phosphate buffered saline. Next, cut a rectangle of PDMS in a thick PDMS slab.Then cut another rectangle inside. In order to have a pool that will retain the cells and medium, add silicone grease under this rectangular cut PDMS and place it on top of the PDMS sheet to create a medium retaining pool. The grease will allow the gliding of the pool over the PDMS sheet during stretching to pattern the cells.First, detach the cells from a culture flask at 50%co fluency. Once the cells are in suspension, pipette them several times with a 200 microliter tip to break up aggregates, which will ensure that individual cells bind to the pattern. Next, add one milliliter of the cell suspension into the pool.Let the cells bind to the patterns for 10 to 30 minutes, depending on the cell. Type in a 37 degree Celsius incubator. Once the cells have attached to the patterns, gently flush the floating cells with equilibrated medium to flush the cells.Remove the medium with an aspirating pipette while simultaneously adding medium. It is critical to maintain sufficient flux to wash away unattached cells while keeping the cells submerged in medium. Next, add a cover slip on top of the pool to avoid evaporation and medium leakage in case the PDMS breaks and allow the cells to spread for a few hours on the patterns.Then put the device on an inverted microscope and start imaging. Avoid the use of oil immersion objectives as they will not work due to focusing problems. To stretch the micro pattern cells turn the micrometric screw while correcting the stage position in the x, y, and Z axes.The stage position needs to be corrected to counteract the widening of the PDMS and the loss of focus. The technique presented in this video protocol allowed the application of forces on the retraction fibers of mitotic mammalian cells by stretching the substrate at the onset of metaphase. Some of these retraction fibers were pulled away from the cell body, resulting in a mechanical force applied on the mitotic cells cortex before stretching, the cell is plated on an oval pattern to achieve this.The patterning is done with the PDMS being stretched during the impression of round patterns through the photo mask. After stretching, the substrate is stretched such that the pattern becomes a circle. After watching this video, you should have a good understanding of how you can plate cells on macro patterns and then apply stretching forces to the substrate.

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

Research

JoVE Journal

Bioengineering

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 Platform for Applications in Cell and Tissue Mechanobiology

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

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.

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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.

Pattern Generation for Micropattern Traction Microscopy

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.

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

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

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Here, we present a versatile equibiaxial stretching device that is pneumatically actuated and compatible with high-resolution microscopy.

Equibiaxial Stretching Device for High Magnification Live-Cell Confocal Fluorescence Microscopy

We present a dynamic and reversible stiffness assay platform fabricated using an ultra-soft silicone elastomer (polydimethylsiloxane, PDMS) with embedded iron particles. This novel assay is suitable for studying emergent time-dependent physical changes in cell phenotypes, including contact guidance. Here, we measure neonatal rat cardiomyocyte alignment upon matrix stiffening using magnetics.

Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

Learn to stretch micropatterned cells on a PDMS membrane for studying cellular responses to mechanical forces. This technique enables controlled force application on adherent cells using video microscopy.

Stretching Micropatterned Cells on a PDMS Membrane

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