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...

<ol>
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A survey of present understanding.</a> J Biomech. 42, 1793-1803 (2009).</li><li>Waters, C. 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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. 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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 border="0" cellpadding="0" cellspacing="0">
	<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>3.5x5.25x0.020&#34;</td>
		</tr>
		<tr>
			<td>Sylgard 184&#160;</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>10:1</td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</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&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>16HBE cells</td>
			<td>ATCC</td>
			<td>CRL-2741</td>
			<td>Human Bronchial Epithelial Cells</td>
		</tr>
		<tr>
			<td>AFM Indentation Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever Beams for Nano-indentation</td>
			<td>Budget Sensors</td>
			<td>Si-Ni30</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite Element Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>Simulia</td>
			<td>6.12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></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 ...

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10.2K vistas.  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. 