The authors would like that thank Fedex Institute of Technology at the University of Memphis for their support. The authors would like to acknowledge students of the senior design project group in the Mechanical Engineering Department at the University of Memphis (David Butler, Jackie Carter, Dominick Cleveland, Jacob Shaffer), Daniel Kohn from the University of Memphis Engineering Technology department for motor control, and Dr. Bin Teng and Ms. Charlean Luellen for their help in cell culture. This work was supported by K01 HL120912 (ER) and R01 HL123540 (CMW).

1. Construction of Membrane with Well Walls for Retention of Cell Culture Media (see Figure 1D for the final product)
<ol>
	<li>Using polydimethylsiloxane (PDMS) sheets coated with Collagen I, cut the outline of the flexible membrane with a scalpel or a die.</li>
	<li>Place each membrane in a 60 mm Petri dish for storage.</li>
	<li>Creation of walls:
	<ol>
		<li>Mix PDMS at a 10:1 weight ratio of elastomer A to elastomer B (curing agent).</li>
		<li>Pour 5 ml of fully mixed PDMS into 50 ml tubes.</li>
		<li>Place 50 ml tubes with uncured PDMS horizontally in a hybridization oven.</li>
		<li>Use the rotor function to coat the inner walls of the tubes at 8 rpm during the curing time. At RT, the PDMS will be fully cured in 2 days.</li>
		<li>Remove the PDMS from the tube in a sterile cell culture hood.</li>
		<li>Using a new razor blade, partition the cylinder of PDMS into sections 4 mm in height.</li>
		<li>Place the sections, which will serve as the membrane walls, into a Petri dish container without allowing the walls to become creased.</li>
	</ol>
	</li>
	<li>Select and center one wall of PDMS on one membrane while maintaining the two in the Petri dish.</li>
	<li>Gently place uncured PDMS (10:1 ratio) on the outside perimeter of the wall to be used as glue. Prevent formation of gaps between the wall and the membrane since it is there to retain liquid.</li>
	<li>Place each Petri dish containing the completed membrane and covered in an oven at 70 &#176;C for 24 hr to cure.</li>
</ol>
2. Correlation of Motor Rotations with Clamp Displacement or Radial Growth for Calibration (Figure 2)
<ol>
	<li>Start the software controlling the motor.</li>
	<li>Displace the clamps of the device using the manual setting in the motor software.</li>
	<li>Measure the distance and record the motor count position between opposing clamps at both minimum and maximum displacement of the clamps.</li>
	<li>Calculate the percent change in the distance between the clamps as a function of the motor count (~0-75k counts). This will indicate the maximum potential strain achieved on the membrane.</li>
</ol>
3. Application of Stretch on Mouse Lung Epithelial Cell Line (MLE12)
<ol>
	<li>Seed MLE12 cells at 2.5 million cells on the flexible membrane (Step 1) per well to be confluent within two days. The seeding density may vary for different cell types.</li>
	<li>Make sure the mechanical actuator is in a fully relaxed position.
	<ol>
		<li>Remove cell culture media.</li>
		<li>Using 1.5 mm biopsy punches, punch two holes in each of the six clamp (see Figure 1D) tabs of the membrane. The radial placement of the holes determines the amount of pre-tension the membrane experiences initially. Punch the holes at a radius of 20.5 mm on the membrane tabs if no pre-tension is desired.</li>
		<li>Position the membrane on the stretcher with the punched holes lining up with the pins within the clamps. Place top clamps in place. Tighten the screws one at a time alternating sides.</li>
		<li>Add 1 ml cell culture media.</li>
	</ol>
	</li>
	<li>Place the device on the microscope stage centering the middle of the membrane with the light path.</li>
	<li>Use tape or magnets (if possible) to fix the device to the stage. Once fixed, control the in-plane (parallel to the membrane) and vertical (z-direction) of the mechanical device with the stage controller of the microscope.</li>
	<li>Apply stretch with the software provided by the manufacturer by manually controlling the position and speed of motor rotation20.</li>
</ol>
4. Measurement of Mitochondrial Reactive Oxygen Species (ROS)
<ol>
	<li>Once cells are confluent, add 1-2 ml of RT DMEM with mitochondrial superoxide indicator (5 &#181;M final concentration) directly on the cells.</li>
	<li>Incubate for 10 min at 37 &#176;C.</li>
	<li>Wash cells gently three times with buffer warmed in a water bath to 37 &#176;C.</li>
	<li>Immediately place the flexible membrane on the stretcher (Step 2.2) already in place under an upright confocal microscope.</li>
	<li>Add 1 ml of DMEM with phenol-red free medium and 25 mM of HEPES.</li>
	<li>Set excitation/emission filters to 510/580 nm.</li>
	<li>Image multiple fields every 15 min to create the desired time course of mitochondrial superoxide production.</li>
	<li>From captured images, record fluorescence intensity histograms at each time interval using software capable of quantifying fluorescence intensity.</li>
</ol>
5. Application of Stretch and Atomic Force Microscopy (AFM)
NOTE: These steps are provided for a specific AFM and optical microscope combination (Figure 4 and Materials List).
<ol>
	<li>Prepare the AFM for the experiment.
	<ol>
		<li>Increase the height of the AFM head to its maximum position in the z-direction.</li>
		<li>Put extenders on the legs of the AFM to lift the plane at which AFM cantilever contacts the sample. The specimen and the AFM head needs to be lifted to accommodate the height of the mechanical device.</li>
	</ol>
	</li>
	<li>Prepare the optical microscope (if available).
	<ol>
		<li>Remove the AFM scanner plate. Remove the desired objective.</li>
		<li>Add a spacer to the objective. The height of the spacer would depend on the objective and the specific AFM set-up, but it is necessary if optical imaging is desired since observation plane will be shift in the z-direction by an amount equal to the stretcher and adapter height (Figure 5A). Note that the AFM usually provides low magnification imaging from an optical path above the device.</li>
		<li>Mount the desired objective back in its location. Place the scanner back on to the AFM.</li>
		<li>Start the AFM software. Start all necessary light sources including the light source for fluorescence measurements.</li>
		<li>Mount a chip with a cantilever beam that is appropriate for the desired measurements. 200 pN/nm or less stiffness is preferred when measuring the elastic modulus of live cells.</li>
		<li>Align the laser and calibrate the cantilever stiffness according to manufacturer&#8217;s suggestions on a glass coverslip mounted on the device.</li>
	</ol>
	</li>
	<li>Prepare a membrane as in Step 1, but with the following modifications.
	<ol>
		<li>Immediately before mounting the membrane on to the stretching device, cut the walls to about 1 mm in height. This prevents the interference experienced between the membrane walls and the load cell.</li>
		<li>Mount the membrane on the mechanical device as described 3.2.1-3.2.3.</li>
		<li>Remove cell culture media to prevent damage to the AFM scanner in case of a spill.</li>
		<li>Couple the device with an adapter (Figure 5A). Place the mechanical device with the adapter on the scanner.</li>
		<li>Stretch the membrane to the desired tensile strain level.</li>
	</ol>
	</li>
	<li>Nano-indentation of stretched cells.
	<ol>
		<li>Add a limited (&#60;0.5 ml) volume of media on the cells to avoid AFM scanner or microscope damage due to a spill.</li>
		<li>Engage the cantilever beam with the membrane.</li>
		<li>Follow the protocol of the particular AFM device to scan areas of interest.&#160;</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Tschumperlin, D. J., Boudreault, F., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+new+opportunities+in+lung+mechanobiology.">Recent advances and new opportunities in lung mechanobiology.</a> J Biomech. 43, 99-107 (2010).</li><li>Waters, C. M., Roan, E., Navajas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comprehensive Physiology. , (2011).</li><li>Majkut, S., Dingal, P. C. D. P., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+Sensitivity+and+Mechanotransduction+during+Heart+Development.">Stress Sensitivity and Mechanotransduction during Heart Development.</a> Current Biology. 24, R495-R501 (2014).</li><li>Hoffman, B. D., Grashoff, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+molecular+processes+mediate+cellular+mechanotransduction.">Dynamic molecular processes mediate cellular mechanotransduction.</a> Nature. 475, 316-323 (2011).</li><li>Wang, N., Butler, J. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+across+the+cell-surface+and+through+the+cytoskeleton.">Mechanotransduction across the cell-surface and through the cytoskeleton.</a> Science. 260, 1124-1127 (1993).</li><li>Liu, M., Tanswell, A. K., Post, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+force-induced+signal+transduction+in+lung+cells.">Mechanical force-induced signal transduction in lung cells.</a> Am J Physiol. 277, L667-L683 (1999).</li><li>Janmey, P. A., McCulloch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+integrating+cell+responses+to+mechanical+stimuli.">Cell mechanics: integrating cell responses to mechanical stimuli.</a> Annu Rev Biomed Eng. 9, 1-34 (2007).</li><li>Waters, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+mechanotransduction.">Reactive oxygen species in mechanotransduction.</a> Am J Physiol Lung Cell Mol Physiol. 287, L484-L485 (2004).</li><li>Chapman, K. E., 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=Cyclic+mechanical+strain+increases+reactive+oxygen+species+production+in+pulmonary+epithelial+cells.">Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 289, L834-L841 (2005).</li><li>Chu, E. K., Whitehead, T., Slutsky, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cyclic+opening+and+closing+at+low-+and+high-volume+ventilation+on+bronchoalveolar+lavage+cytokines.">Effects of cyclic opening and closing at low- and high-volume ventilation on bronchoalveolar lavage cytokines.</a> Crit Car Med. 32, 168-174 (2004).</li><li>Tschumperlin, D., Margulies, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equibiaxial+deformation-induced+injury+of+alveolar+epithelial+cells+in+vitro.">Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro.</a> Am J Physiol. 275, L1173-L1183 (1998).</li><li>Vlahakis, N. E., Hubmayr, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+failure+in+ventilator-injured+lungs.">Cellular stress failure in ventilator-injured lungs.</a> Am J Respir Crit Care Med. 171, 1328-1342 (2005).</li><li>Roan, E., 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=Hyperoxia+alters+the+mechanical+properties+of+alveolar+epithelial+cells.">Hyperoxia alters the mechanical properties of alveolar epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 302, L1235-L1241 (2012).</li><li>Gamerdinger, K., 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+load+and+mechanical+integrity+of+lung+cells+-+Experimental+mechanostimulation+of+epithelial+cell-+and+fibroblast-monolayers.">Mechanical load and mechanical integrity of lung cells - Experimental mechanostimulation of epithelial cell- and fibroblast-monolayers.</a> J Mech Behav Biomed Mater. 4, 201-209 (2014).</li><li>Hayashi, K., Naiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+remodeling+of+vascular+wall;+biomechanical+response+to+hypertension.">Adaptation and remodeling of vascular wall; biomechanical response to hypertension.</a> J Mech Behav Biomed Mater. 2, 3-19 (2009).</li><li>Villemure, I., Stokes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+plate+mechanics+and+mechanobiology.+A+survey+of+present+understanding.">Growth plate mechanics and mechanobiology. A survey of present understanding.</a> J Biomech. 42, 1793-1803 (2009).</li><li>Waters, 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=A+system+to+impose+prescribed+homogenous+strains+on+cultured+cells.">A system to impose prescribed homogenous strains on cultured cells.</a> J Appl Physiol (1985). 91, 1600-1610 (2001).</li><li>Gerstmair, A., Fois, G., Innerbichler, S., Dietl, P., Felder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+for+simultaneous+live+cell+imaging+during+uni-axial+mechanical+strain+or+compression.">A device for simultaneous live cell imaging during uni-axial mechanical strain or compression.</a> J Appl Physiol (1985). 107, 613-620 (1985).</li><li>Dassow, C., 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+method+to+measure+mechanical+properties+of+pulmonary+epithelial+cell+layers.">A method to measure mechanical properties of pulmonary epithelial cell layers.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 101, 1164-1171 (2013).</li><li>Chapman, K., 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=Cyclic+mechanical+strain+increases+reactive+oxygen+species+production+in+pulmonary+epithelial+cells.">Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 289, L834-L841 (2005).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+stretch,+reactive+oxygen+species,+and+vascular+remodeling.">Cyclic stretch, reactive oxygen species, and vascular remodeling.</a> Antioxid Redox Signal. 11, 1651-1667 (2009).</li><li>Turrens, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+formation+of+reactive+oxygen+species.">Mitochondrial formation of reactive oxygen species.</a> J Physiol. 552, 335-344 (2003).</li><li>Wang, W., 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=Superoxide+flashes+in+single+mitochondria.">Superoxide flashes in single mitochondria.</a> Cell. 134, 279-290 (2008).</li><li>Pouvreau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+flashes+in+mouse+skeletal+muscle+are+produced+by+discrete+arrays+of+active+mitochondria+operating+coherently.">Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently.</a> PLoS One. 5, (2010).</li><li>Yalcin, H. C., 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=Influence+of+cytoskeletal+structure+and+mechanics+on+epithelial+cell+injury+during+cyclic+airway+reopening.">Influence of cytoskeletal structure and mechanics on epithelial cell injury during cyclic airway reopening.</a> Am J Physiol Lung Cell Mol Physiol. 297, L881-L891 (2009).</li><li>Jacob, A. M., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atelectrauma+disrupts+pulmonary+epithelial+barrier+integrity+and+alters+the+distribution+of+tight+junction+proteins+ZO-1+and+claudin+4.">Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4.</a> J Appl Physiol. 113, 1377-1387 (2012).</li><li>DiPaolo, B. C., Lenormand, G., Fredberg, J. J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+magnitude+and+frequency-dependent+actin+cytoskeleton+remodeling+in+alveolar+epithelia.">Stretch magnitude and frequency-dependent actin cytoskeleton remodeling in alveolar epithelia.</a> Am J Physiol Cell Physiol. 299, C345-C353 (2010).</li></ol>

The authors declare that they have no competing financial interests.

A unique device for live cell imaging during mechanical stretch was developed; and this device was used in a protocol to study lung epithelial cell mechanobiology. In preliminary studies, it was found that a single held stretch stimulated the production of mitochondrial superoxide in bronchial epithelial cells. In addition, it was demonstrated that increased levels of mechanical strain caused direct damage to the integrity of a monolayer of alveolar epithelial cells.
To conduct these prelimina...

Cells are subjected to mechanical loads in many tissues, and this mechanical stimulation has been shown to promote changes in patterns of gene expression, release of growth factors, cytokines, or remodeling of the extracellular matrix and cytoskeleton1-4. The intracellular signals transduced from such mechanical stimuli occur through the process of mechanotransduction5-7. In the respiratory system, one outcome of mechanotransduction is the increase in reactive oxygen species (ROS)8,9 and pro-inflammatory cytokines10 in pulmonary epithelial cells in the presence of cyclic tensile strain. Strong evidence also suggests that excessive tensile strain leads to direct injury to the alveolar epithelium, in addition to the biochemical responses of cells11-14. Although the focus here is primarily on the response of lung cells to mechanical deformation, pathways induced by mechanotransduction play a key role in the basic function of many tissues in the human body, including the regulation of vascular tone15 and the development of the growth plate16.

The growing interest in mechanotransduction has resulted in the development of numerous devices for the application of physiologically relevant mechanical loads to cultured cells and tissue. In particular, devices applying a tensile strain, which is a common form of mechanical loading experienced by tissue, are popular 11,17-19. However, many of the available devices are either designed as a bioreactor for tissue engineering applications or are not conducive to real time imaging with stretch. As such, there is a need to develop tools and methods that can visualize cells and tissues in tension to facilitate the investigation of pathways of mechanotransduction.
Herein, an in-plane mechanical stretching device was designed and protocols were developed to apply multiple forms of strain to tissues and cells while allowing imaging of the biochemical and mechanical responses in real time (Figure 1A-D). The device utilizes six evenly spaced clamps arranged circumferentially to grasp a flexible membrane and apply an in-plane, radial distention up to approximately 20% (Figure 1B). The actuating device can be placed in a cell culture incubator for an extended period of time, while the motor (Figure 1C) is positioned outside the incubator and controlled by proprietary software provided by the motor supplier. The motor is connected to a linear driver, which rotates an internal cam, driving the six stretcher clamps uniformly in tension and relaxation.
In addition to the mechanical device, customized flexible membranes were created from commercially available cell culture ready membranes to be used in the mechanical system. Then circular walls (with a diameter of approximately 28 mm) were made and attached on to the flexible membrane so that cells could be cultured only in this region of well-described strain profile. In order to determine whether the placement of these membranes within the actuating device would provide uniform and isotropic strain in the center of the flexible membrane, finite element analysis was conducted using commercially available software (Figure 1E-F). The flexible membrane was modeled with symmetric boundary conditions and utilizing all quadrilateral elements for the mesh. The concentric rings seen in the contour plot of maximum principal strain shown in Figure 1F indicate the isotropic distribution of the strain.
The strain experienced by the membrane was measured by recording images of markings through loading (Figure 2). Figure 2D shows that the average membrane strain measured in radial and axial directions was approximately linear with respect to the applied motor counts up to a maximum linear strain of 20%. There was no significant difference between the strain levels measured during distention compared with those measured during retraction back to the resting position. Next, the displacement of human bronchial epithelial cells (16HBE) and their nuclei cultured on the custom flexible membrane were measured. Fluorescently labeled (DAPI) nuclei of the 16HBE cells were imaged using a 20X objective under a confocal microscope, whereas whole cell displacement was measured with phase contrast images recorded with a digital microscope. As seen in Figure 3, the strain measured by displacement of nuclei was similar to that measured by displacement of markings on the membrane, up to ~20% linear strain. This confirms that the strain applied to the membranes was transmitted to the adherent cells. The protocols describing the use of the custom device on a traditional microscope and an atomic force microscope are provided in the following steps.

<table><tbody><tr><td>SmartMotor NEMA 34: 3400 Series</td><td>MOOG Animatics</td><td>SM3416D</td><td>Integrated motor, controller, amplifier, encoder and communications bus</td></tr><tr><td>Flexcell Membrane (Collagen I coated)</td><td>Flexcell International Corp</td><td>SM2-1010C</td><td>3.5&quot;&amp;nbsp;x&amp;nbsp;5.25&quot;&amp;nbsp;x&amp;nbsp;0.020&quot;</td></tr><tr><td>Sylgard 184</td><td>Dow Corning Corporation</td><td></td><td>10:1</td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H1399</td><td>DAPI stain</td></tr><tr><td>MitoSOX</td><td>Sigma-Aldrich</td><td>M36008</td><td></td></tr><tr><td>Tiron</td><td>Sigma-Aldrich</td><td>D7389&amp;nbsp;</td><td>mitochondrial superoxide label</td></tr><tr><td>DMEM</td><td></td><td></td><td>superoxide inhibitor</td></tr><tr><td>FBS</td><td></td><td></td><td></td></tr><tr><td>HEPES</td><td></td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>Fisher Scientific</td><td>06-443-19</td><td>Any centriguge tube can be used to create an area for imaging.</td></tr><tr><td>Hybridization oven</td><td>Bellco Glass</td><td></td><td></td></tr><tr><td>MLE12 Cells</td><td>ATCC</td><td>CRL-2110</td><td>Mouse Lung Epithelial Cells&amp;nbsp;</td></tr><tr><td>16HBE cells</td><td>ATCC</td><td>CRL-2741</td><td>Human Bronchial Epithelial Cells</td></tr><tr><td colspan="4">&lt;strong&gt;AFM Indentation Experiments&lt;/strong&gt;</td></tr><tr><td>Cantilever Beams for Nano-indentation</td><td>Budget Sensors</td><td>Si-Ni30</td><td></td></tr><tr><td>AFM&amp;nbsp;</td><td>Asylum Research</td><td>MFP3D</td><td></td></tr><tr><td>Olympus microscope</td><td>Olympus</td><td>IX-71</td><td>Inverted microscope with 20X and 40X objectives.</td></tr><tr><td>AFM Leg Extenders</td><td>Asylum Research</td><td>Not available</td><td>AFM microscope</td></tr><tr><td colspan="4">&lt;strong&gt;Finite Element Analyses&lt;/strong&gt;</td></tr><tr><td>ABAQUS</td><td>Simulia</td><td>6.12</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Microscopes&lt;/strong&gt;</td></tr><tr><td>Digital microscope</td><td>Life Technologies</td><td>EVOS XL Core</td><td>Initially a self standing company, now owned by Life Technologies.</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 710</td><td>2-photon upright microscope</td></tr></tbody></table>

live cell imaging during mechanical stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

Reactive Oxygen Species and Deformation
Previous studies have shown increases in reactive oxygen species (ROS) in airway and alveolar epithelial cells in response to cyclic stretch21. Reactive oxygen species include molecules and free radicals derived from molecular oxygen with high reactivity to lipids, proteins, polysaccharides, and nucleic acids22-24. ROS serve as a common intracellular signal to regulate ion channel function, protein kinase/phosphatase activa...

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

live-cell-imaging-during-mechanical-stretch

Research

JoVE Journal

Bioengineering

10.3K Views.  University of Tennessee Health Science Center. A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy. 

Video: Live Cell Imaging during Mechanical Stretch

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Biology

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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.

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 ...

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.

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

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Neuroscience

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...