Name: a novel stretching platform for applications in cell and tissue mechanobiology
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DT was supported by a postdoctoral studentship from le Fonds de Recherche du Qu&#233;bec-Nature et Technologies (FQRNT) and a MITACS Elevate Strategic Fellowship. CMC was supported by a postdoctoral studentship from le Fonds de Recherche en Sant&#233; du Qu&#233;bec (FRSQ) and the Ernest and Margaret Ford cardiology endowed research fellowship from the University of Ottawa Heart Institute. EOB was supported by operating grants MOP80204 from the Canadian Institute for Health Research (CIHR) and T6335 from the Heart and Stroke Foundation of Ontario. The CIHR and Medtronic collectively provide EOB with a peer-reviewed Research Chair (URC #57093). AEP is funded by the Natural Sciences and Engineering Research Council (NSERC) Discovery Grant, an NSERC Discovery Accelerator Supplement and gratefully acknowledges the support of the Canada Research Chairs (CRC) program and an Early Researcher Award from the Province of Ontario.

1. Mechanical Deformation of Single Cells
<ol>
	<li>Fabrication of a PDMS Substrate with Embedded Fluorescent Beads 
	Prior to fabrication of the substrate, fluorescent microspheres in water solution are resuspended in isopropanol to enhance bead mixing in the PDMS due to its hydrophobic nature.
	<ol>
		<li>Pipette 500 &#956;l of fluorescent microspheres into a 1.5 ml microcentrifuge tube and centrifuge at 16,200 x g for 10 min.</li>
		<li>Discard the supernatant and add 500 &#956;l of isopropanol followed with 5 ...

<ol>
	<li>Yim, E. K., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-dependent+cell+signaling+in+stem+cell+differentiation.">Force-dependent cell signaling in stem cell differentiation.</a> Stem Cell Res Ther. 3, (2012).</li><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>Wang, N., Tytell, J. D., Ingber, D. 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M., Ye, J., Bellin, R., LeDuc, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+and+shear+flow+induced+reorganization+and+recruitment+of+fibronectin+in+fibroblasts.">Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts.</a> Sci Rep. 1, (2011).</li><li>Wang, D., Xie, Y., Yuan, B., Xu, J., Gong, P., Jiang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stretching+device+for+imaging+real-time+molecular+dynamics+of+live+cells+adhering+to+elastic+membranes+on+inverted+microscopes+during+the+entire+process+of+the+stretch.">A stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire process of the stretch.</a> Integr Biol (Camb). 2, 288-293 (2010).</li><li>Haskett, D., Johnson, G., Zhou, A., Utzinger, U., Van de Geest, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructural+and+biomechanical+alterations+of+the+human+aorta+as+a+function+of+age+and+location.">Microstructural and biomechanical alterations of the human aorta as a function of age and location.</a> Biomech Model Mechanobiol. 9, 725-736 (2010).</li><li>Duprey, A., Khanafer, K., Schlicht, M., Avril, S., Williams, D., Berguer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterisation+of+physiological+and+maximum+elastic+modulus+of+ascending+thoracic+aortic+aneurysms+using+uniaxial+tensile+testing.">In vitro characterisation of physiological and maximum elastic modulus of ascending thoracic aortic aneurysms using uniaxial tensile testing.</a> Eur J Vasc Endovasc Surg. 39, 700-707 (2010).</li><li>Tremblay, 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=A+comparison+of+mechanical+properties+of+materials+used+in+aortic+arch+reconstruction.">A comparison of mechanical properties of materials used in aortic arch reconstruction.</a> Ann Thorac Surg. 88, 1484-1491 (2009).</li><li>Khanafer, K., Duprey, A., Zainal, M., Schlicht, M., Williams, D., Berguer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+elastic+modulus+of+ascending+thoracic+aortic+aneurysm+at+different+ranges+of+pressure+using+uniaxial+tensile+testing.">Determination of the elastic modulus of ascending thoracic aortic aneurysm at different ranges of pressure using uniaxial tensile testing.</a> The Journal of thoracic and cardiovascular surgery. 142, 682-686 (2011).</li><li>Van de Geest, J. P., Sacks, M. S., Vorp, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+aneurysm+on+the+biaxial+mechanical+behavior+of+human+abdominal+aorta.">The effects of aneurysm on the biaxial mechanical behavior of human abdominal aorta.</a> J Biomech. 39, 1324-1334 (2006).</li><li>Guolla, L., Bertrand, M., Haase, K., Pelling, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+transduction+and+strain+dynamics+in+actin+stress+fibres+in+response+to+nanonewton+forces.">Force transduction and strain dynamics in actin stress fibres in response to nanonewton forces.</a> J Cell Sci. 125, 603-613 (2012).</li><li>Wang, J. H., Goldschmidt-Clermont, P., Wille, J., Yin, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+endothelial+cell+reorientation+in+response+to+cyclic+mechanical+stretching.">Specificity of endothelial cell reorientation in response to cyclic mechanical stretching.</a> J Biomech. 34, 1563-1572 (2001).</li><li>Jungbauer, S., Gao, H., Spatz, J. P., Kemkemer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+characteristic+regimes+in+frequency-dependent+dynamic+reorientation+of+fibroblasts+on+cyclically+stretched+substrates.">Two characteristic regimes in frequency-dependent dynamic reorientation of fibroblasts on cyclically stretched substrates.</a> Biophys J. 95, 3470-3478 (2008).</li><li>Dahl, K. N., Ribeiro, A. J. S., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+shape,+mechanics,+and+mechanotransduction.">Nuclear shape, mechanics, and mechanotransduction.</a> Circ Res. 102, 1307-1318 (2008).</li><li>Shivashankar, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosignaling+to+the+cell+nucleus+and+gene+regulation.">Mechanosignaling to the cell nucleus and gene regulation.</a> Annu Rev Biophys. 40, 361-378 (2011).</li><li>Chiquet, M., Gelman, L., Lutz, R., Maier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mechanotransduction+to+extracellular+matrix+gene+expression+in+fibroblasts.">From mechanotransduction to extracellular matrix gene expression in fibroblasts.</a> Biochim Biophys Acta. 1793, 911-920 (2009).</li><li>Sullivan, T., 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=Loss+of+A-type+lamin+expression+compromises+nuclear+envelope+integrity+leading+to+muscular+dystrophy.">Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy.</a> J Cell Biol. 147, 913-920 (1999).</li><li>Lammerding, 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=Lamin+A/C+deficiency+causes+defective+nuclear+mechanics+and+mechanotransduction.">Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction.</a> J Clin Invest. 113, 370-378 (2004).</li><li>Tremblay, D., Andrzejewski, L., Leclerc, A., Pelling, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+microtubules+play+distinct+roles+in+governing+the+anisotropic+deformation+of+cell+nuclei+in+response+to+substrate+strain.">Actin and microtubules play distinct roles in governing the anisotropic deformation of cell nuclei in response to substrate strain.</a> Cytoskeleton. , (2013).</li><li>Cuerrier, C. M., 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=Chronic+over-expression+of+heat+shock+protein+27+attenuates+atherogenesis+and+enhances+plaque+remodeling:+a+combined+histological+and+mechanical+assessment+of+aortic+lesions.">Chronic over-expression of heat shock protein 27 attenuates atherogenesis and enhances plaque remodeling: a combined histological and mechanical assessment of aortic lesions.</a> PLoS ONE. 8, (2013).</li><li>Tremblay, D., Cartier, R., Mongrain, R., Leask, R. 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The BAXS platform presented here facilitates numerous experiments in the study of mechanobiology, from investigations of single cells to whole tissues. In addition, the platform is highly flexible and configurable, allowing for numerous mechanical stimulation experiments and multi-axial tensile testing. The platform also enables the maintenance of cells and tissues in physiological conditions and allows for simultaneous microscopy during stretching experiments. The two experiments described in the previous sections demon...

The mechanical microenvironment plays an important role in many cell functions such as proliferation, migration, and differentiation, which have a profound impact in the development and homeostasis of tissues and also in diseases1-6. Over the years, a multitude of experimental tools have been used to mechanically stimulate cells or tissues and measure mechanical properties of biological tissues with the goal of increasing our understanding of basic mechanobiology and studying the onset and progression of diseases6-17. However, one must often rely on several different experimental devices in order to achieve the goals of a particular study. This article presents a single, multi-functional, biaxial stretching (BAXS) platform that allows for studies that investigate the role that mechanical properties and mechanical forces play in biology at the sub-cellular to whole tissue length scales. The BAXS platform not only allows for the quantification of the mechanical properties of isolated tissues, but also facilitates the ability to apply simple, complex, and dynamic strain fields to living cells in order to understand their responses to stretching that occurs in vivo. The BAXS platform also maintains the capacity to perform live-cell microscopy during mechanical testing and perturbations on cells and tissues.
The BAXS platform is a custom-built apparatus that can be used to investigate the effect of substrate deformation at the cellular level and perform tensile tests on biological tissues (Figure 1A). An aluminum heater was fabricated to accommodate a standard 10-cm Petri dish and maintain any physiological solutions at 37 &#176;C using a temperature controller and kapton heaters (Figure 1B). This BAXS platform can be integrated onto an inverted phase contrast and/or fluorescence microscope and allows for simultaneous imaging (Figure 1C). In brief, the BAXS platform consists of four linear voice coil motors of which the moving parts are mounted on miniature linear motion ball bearing slides oriented along two perpendicular axes (Figure 1D). A linear positioning stage is mounted to each of the four motors to allow vertical movement of the clamping system that will be used (Figure 1E). The position of each motor is monitored by an optical encoder with a resolution of 500 nm (Figure 1F). All four motors are independently controlled with a motion controller employing optical encoder feedback to execute motion commands (Figure 1G). A LabVIEW interface provides full control over the displacement magnitude, speed, and acceleration of each motor in order to generate completely customizable, static and dynamic, deformation of the cells or tissue samples.
The technique used to induce a deformation in cells is achieved by simply allowing cells to firmly adhere to a flexible and transparent substrate and then stretching this substrate using the four motors of the BAXS platform. The BAXS platform allows mounting of any custom-designed set of clamps to attach the substrate on the voice coil motors. For this purpose, we designed a set of clamps to which a flexible and transparent substrate, made of polydimethylsiloxane (PDMS), can be attached (Figures 2A-C and Figure 3). As the clamps will be exposed to physiological solutions, all parts were machined from stainless steel to allow for sterilization. These clamps have been carefully designed to bring the substrate as close as possible to the microscope objective to enhance image quality while minimizing the stress on the substrate during stretching (Figure 2D).
The same BAXS platform can also be used to quantify the stiffness of small tissue samples, using an appropriate set of clamps with adapted supports for the tissue samples and a load cell to monitor forces. Several approaches can be taken to mount a tissue to the BAXS platform motors; in this case the stainless steel insect minutiens pins can hook through the opening of vascular tissues in order to perform tensile tests (Figures 4A-B). Alternatively, for thick tissues without a natural opening, tissue edges can either be held in position with the clamps attached to the voice coil motors or glued to small glass slides with biological glue and attached to the motors with the clamps. In order to perform tensile tests a miniature load cell is required and can be easily incorporated onto the BAXS platform motors and used to measure the force acting on the tissue during a stretching cycle (Figure 4C). As the BAXS platform is composed of four motors, the introduction of a second load cell allows one to perform tensile testing along two orthogonal directions. This ability allows one to quantify the mechanical stiffness of a single tissue along two perpendicular directions during the same experiment.
Importantly, in all configurations, the cells or tissue samples of interest are always maintained in a temperature-controlled bath that is accessible to the user. This ability allows for the introduction of pharmacological agents during sample stretching in order to examine the temporal response of the sample. Additionally, as the optical axis of the inverted microscope remains unobstructed, all forms of microscopy are still available to the user. Finally, as all four motors of the BAXS platform are independent it is possible to apply highly configurable strain fields to the sample of interest. In vivo cells and tissues are exposed to complex and anisotropic stretching that can be more appropriately mimicked in this platform as opposed to traditional uniaxial stretching platform7,13,15,18,19. Moreover, the physical characteristics of the strain field can be changed on the fly during an experiment. These abilities allow the user to examine the cellular and tissue level response to a wide number of highly complex, anisotropic, temporally, and spatially varying strain fields. This article describes the advantages and limitations of the BAXS platform as well as its design, operating principles, and the experimental details for single cell and whole tissue experiments.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig1highres.jpg" width="500" /> 
Figure 1.&#160;Overview of the BAXS platform. A) Top view of the BAXS platform showing the four voice coil motors. B) Detailed picture of the Petri dish heater used to maintain cells and tissues at 37 &#176;C. C) The platform can be mounted on an inverted microscope to perform live-cell imaging during stretching experiments. D) Detailed picture of the voice coil motor; the moving part of the platform. E) Detailed picture of the linear positioning stage allowing vertical displacement of the clamping systems. F) Detailed picture of the optical encoder that provides real-time position of the motor to the motion controller. G) Detailed picture of the motion controller showing the four optical encoder inputs and power outputs to the four voice coil motors.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig2highres.jpg" width="500" /> 
Figure 2.&#160;Clamping system for cell stretching experiments. A-B) Pictures showing the details of the clamps used to attach the PDMS substrate to the voice coil motors for stretching. C) The substrate is wrapped around the cylindrical part of the clamp with its anchoring features sitting into the groove at the top. Then the substrate is secured using the setscrews that push the substrate/anchoring features into the top groove. D) Illustration of the BAXS platform with the clamps holding the substrate in place. The inset shows a detailed view of the substrate with cells attached to it sitting just above a cover slip and the microscope objective.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig3highres.jpg" width="500" /> 
Figure 3.&#160;Bill of materials of the membrane and its clamping system. Drawings showing the dimensions of the principal parts integrated to the biaxial platform to perform cell stretching experiments.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig4highres.jpg" width="500" /> 
Figure 4.&#160;Example of a clamping system for stiffness assessment of small caliber vessels. A-B) Detailed pictures of the clamping system used to induce deformation in a 1 mm diameter mouse aorta. Stainless steel pins were carefully shaped into open triangles to allow the vessel to slide on both pins. C) Illustration of the BAXS platform with the clamps holding the vessel and a load cell attached between the fixed motor and the left clamp. The inset shows a detailed top-view of the vessel mounted on the pins.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PDMS</td>
			<td style="width:155px;">Ellsworth Adhesives</td>
			<td style="width:255px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:167px;">The ratio base to cross-linker used in this protocol is 20:1. Mix in a laminar hood to keep dust from contamining your&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">FluoSpheres fluorescent microspheres</td>
			<td style="width:155px;">Invitrogen</td>
			<td style="width:255px;">F8810</td>
			<td>Keep away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Linear voice coil</td>
			<td style="width:155px;">Moticont</td>
			<td style="width:255px;">LVCM-051-051-01</td>
			<td>The motro comes in two pieces (magnet and coil). It has to be mounted on a ball bearing sytem to be functional.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Ball bearing slide</td>
			<td style="width:155px;">Edmund Optics</td>
			<td style="width:255px;">NT37-360</td>
			<td>Miniature and Small Linear Motion Ball Bearing Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">linear positioning stage</td>
			<td style="width:155px;">Edmund Optics</td>
			<td style="width:255px;">38-960</td>
			<td>Center Drive 1.25&#34; Square Linear Translation Stages</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">optical encoder</td>
			<td style="width:155px;">GSI microE systems</td>
			<td style="width:255px;">Mercury II 1600S - 0.5um resolution</td>
			<td>reflective incremental encoder.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">motion controller</td>
			<td style="width:155px;">Galil</td>
			<td style="width:255px;">DMC-2143(DIN)-DC48 with AMP-20440</td>
			<td>4 axis controller with a 4 axis amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">load cell&#160;</td>
			<td style="width:155px;">Honeywell</td>
			<td style="width:255px;">31 low</td>
			<td>miniature load cell with a range of 0-150 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Insect minutiens pins (0,20mm)</td>
			<td style="width:155px;">Pin Service Austerlitz Insect pins</td>
			<td style="width:255px;"></td>
			<td>Stainless steel pins that are bended in an opened triangle shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">SU-8 2050</td>
			<td style="width:155px;">Micro Chem</td>
			<td style="width:255px;">SU-8 2050</td>
			<td>Permanent epoxy negative photoresist. Keep away from heat and light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Air-plasma treatment system</td>
			<td style="width:155px;">Glowresearch</td>
			<td style="width:255px;"></td>
			<td>Autoglow Oxygen Plasma System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Rat-tail collagen</td>
			<td style="width:155px;">Invitrogen</td>
			<td style="width:255px;">A10483-01</td>
			<td>Collagen I, Rat Tail 5mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Hoechst 33342</td>
			<td style="width:155px;">Invitrogen</td>
			<td style="width:255px;">R37605</td>
			<td>DNA-specific fluorescent dye. Keep in the fridge.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Kapton (Polyimide Film) Insulated Flexible Heaters</td>
			<td style="width:155px;">omega.ca</td>
			<td style="width:255px;">KHLV-0504/(10)-P</td>
			<td>28V flexible heaters; can be supplied with a 24V</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">1/16 DIN Autotune Temperature and Process Controllers</td>
			<td style="width:155px;">omega.ca</td>
			<td style="width:255px;">CN63200-R1-LV</td>
			<td>Temperature controller; supply 24 V.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">DMEM culture medium</td>
			<td style="width:155px;">Hyclone</td>
			<td style="width:255px;">SH3024301</td>
			<td>Dulbecco&#8217;s Modified 30 Eagle Medium. Keep at 4 C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Penicillin-Streptomycin</td>
			<td style="width:155px;">Hyclone</td>
			<td style="width:255px;">SV30010</td>
			<td>Keep stock frozen. Keep working solution at 4 C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fetal bovine serum (FBS)</td>
			<td style="width:155px;">Hyclone</td>
			<td style="width:255px;">SH3039603C</td>
			<td>Keep frozen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Trypsin 0,05%</td>
			<td style="width:155px;">Hyclone</td>
			<td style="width:255px;">SH30236,02</td>
			<td>Keep frozen. Digestion of cell attachement proteins for subcultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Hepes</td>
			<td style="width:155px;">Wisent Inc</td>
			<td style="width:255px;">330-050-EL</td>
			<td>HEPES-buffered salt solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">NaCl</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">BP358-1</td>
			<td>HEPES-buffered salt solution / Krebs physiological solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">KCl</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">BP366-500</td>
			<td>HEPES-buffered salt solution / Krebs physiological solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:273px;">MgSO4</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">M65-500</td>
			<td>HEPES-buffered salt solution / Krebs physiological solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:273px;">CaCl2</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">C614-500</td>
			<td>HEPES-buffered salt solution / Krebs physiological solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dextrose</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">BP220-1</td>
			<td>HEPES-buffered salt solution / Krebs physiological solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:273px;">NaHCO3</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">BP328-1</td>
			<td>Krebs physiological solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:273px;">KH2PO4</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:255px;">BP362-500</td>
			<td>Krebs physiological solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:273px;">Carbogen 95% O2/ 5% CO2</td>
			<td style="width:155px;">Lindle</td>
			<td style="width:255px;">DIN:02154749</td>
			<td>Krebs physiological solution oxygenation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Nocodazole</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:255px;">M1404</td>
			<td>Microtubules depolymerization agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cytochalasin-D</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:255px;">C8273</td>
			<td>Actin filaments depolymerization agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-&#945;-SMA-FITC</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:255px;">F3777</td>
			<td>Used to stain and quantify smooth muscle cells content</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Picrosirius red stain</td>
			<td style="width:155px;">Fluka</td>
			<td style="width:255px;">43665</td>
			<td>Used to stain and quantify collagen content</td>
		</tr>
	</tbody>
</table>

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.

Cell Stretching
The BAXS platform was used to investigate the mechanical response of the nucleus in single mouse myoblast cells (C2C12) exposed to a substrate deformation of 25%. Myoblast cells are found in muscle tissue and are constantly exposed to mechanical stretch and compression in vivo. The shape and mechanical properties of the cell nucleus have shown to play a major role in the regulation of gene expression and transcriptional activity20,21 and als...

Watch this Scientific Journal Video about A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology at JoVE.com

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

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.

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

Research

JoVE Journal

Bioengineering

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