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 min of vortexing. Put the vial aside overnight in the dark in order to allow any large particles aggregates to sediment.</li>
		<li>The next morning, carefully remove the supernatant to a clean microcentrifuge vial. This bead solution can be used to fabricate more than 5 substrates. NOTE: The bead solution will continue to sediment for the next 3 days. Be careful to avoid resuspending the pellet.</li>
		<li>Pour 0.5 g of the curing agent provided with the PDMS kit in a 1.5 ml microcentrifuge vial using a scientific balance. By successive steps, add a total of 90 &#956;l of beads (in six 15 &#956;l additions), vortexing for 1 min between each addition. Set aside.</li>
		<li>Weigh 10 g of PDMS and mix for at least 12 min with the 0.5 g of the curing agent supplemented with fluorescent beads.</li>
		<li>Fabricate an SU-8 2050 cross-shaped mold using standard photolithography techniques following manufacturer&#8217;s instructions. The mold used has a height of 320 &#956;m and an area of 13.4 cm2 (Figure 3). The mold can contain 428 &#956;l or 440 mg of PDMS.</li>
		<li>Pour 400 mg of the PDMS with beads in the cross-shaped mold using a transfer pipette and cure for 2 hr at 80 &#176;C. After curing, peel off the substrate from the mold (Figure 5A). The substrate can be kept in a Petri dish at room temperature for 2 weeks without showing significant changes in its mechanical properties.</li>
		<li>Pour droplets of PDMS (curing agent:PDMS with a ratio of 1:20) in a Petri dish with a final size of approximately 4 mm in diameter and cure them upside down for 2 hr at 80 &#176;C (Figure 5B). These anchoring features can be kept in a Petri dish for weeks. NOTE: Maintain the dish upside down to prevent the drops from flattening during the curing process.</li>
		<li>Air-plasma treat (30 sec at 30 W) the substrate and 8 anchoring features. Bind the features on each end of the substrate at a distance of 4 mm from the square shaped indent present on the substrate (Figure 5C).</li>
	</ol>
	</li>
	<li>Mounting Membrane on the Clamps
	<ol>
		<li>Wrap each end of the substrate around the grooved cylindrical part of the clamps and secure it in place with the 2&#160;setscrews from the top (Figure 2B and Figures 5D-E).</li>
		<li>Screw the 4&#160;clamps on the clamp holder and pour PDMS (ratio 1:20) using a disposable transfer pipette at the interface between the substrate and the grooved cylindrical part of the clamps. Spread the uncured PDMS around the grooved cylindrical part using a 1.5 mm hex key.</li>
		<li>Pour PDMS (1:20) in the grooves until completely filled by capillary action and cure the assembly at 80 &#176;C for 2 hr (Figure 5F).</li>
	</ol>
	</li>
	<li>Seeding Cells on the Membrane
	<ol>
		<li>Air-plasma treat (30 sec at 30 W) the whole assembly to sterilize and functionalize the substrate to allow for collagen coating.</li>
		<li>Functionalize the area of the substrate where cells will be seeded with 1 ml of 0.02 M acetic acid supplemented with 16 &#956;g/ml of rat-tail collagen at room temperature for 1 hr. The desired final collagen density is 5 &#956;g/cm2.</li>
		<li>Rinse the substrate 3x with phosphate buffer and let it dry at room temperature for at least for 10 min.</li>
		<li>Add 40 &#956;l of culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin containing 2,000 cells in the center portion of the substrate to cover an area of 1 cm2 (cell density: 20 cells/mm2). Cell density can be altered according to experimental requirements.</li>
		<li>Put the whole assembly in a standard cell culture incubator with the substrate facing up with the drop of culture medium containing cells on it. NOTE: The assembly must be kept with the substrate facing up for at least 3 hr to allow cells to firmly attach it. To prevent evaporation, 30 &#956;l of warm culture medium is added into the drop on the substrate every 45 min for 3 hr.</li>
		<li>After 3 hr, flip the whole assembly into a Petri dish filled with fresh culture medium to submerge the substrate and incubate overnight to allow cells to proliferate.</li>
		<li>The next day, prepare HEPES-buffered salt solution (HBSS; 20 mM of Hepes, 120 mM of NaCl, 5.3 mM of KCl, 0.8 mM of MgSO4, 1.8 mM of CaCl2, and 11.1 mM of dextrose). Adjust the pH to 7.4. NOTE: The HBSS solution has to be prepared daily and kept at 37 &#176;C during the experiments. This physiological solution is used to maintain the cells on the microscope stage by mimicking the normal tissue/blood environment.</li>
		<li>Mount the set-up on inverted phase contrast or fluorescent microscope Mount the clamp-substrate assembly on the BAXS platform and motors. Fill the Petri dish inside the Petri dish heater with HEPES buffer (Figure 2D).</li>
	</ol>
	</li>
</ol>
2. Stiffness Measurement of Small Caliber Vessels
<ol>
	<li>Preparation
	<ol>
		<li>Krebs physiological solution: Prepare a solution of 118.1 mM NaCl, 11.1 mM D-glucose, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 2.5 mM CaCl2. Adjust the pH to 7.4 and oxygenate the solution with carbogen medical gas (95% O2/5% CO2) for 30 min. NOTE: The Krebs solution has to be prepared daily and kept at 37 &#176;C during the experiments. This physiological solution is used to maintain the tissues alive by mimicking the normal tissue/blood environment.</li>
		<li>Gather instrumentation for the dissection and mechanical assessment of aortic vessels: surgical scissors, bended tweezers, micro-scissors, surgical dissecting microscope, 50 ml polypropylene centrifuge tubes, and 10 ml serological pipettes. The surgical and experiment procedure do not require any sterile conditions. Mount the clamps of the BAXS platform along with the load cell beforehand.</li>
	</ol>
	</li>
	<li>Tissue Isolation and Dissection 
	All experimental procedures involving laboratory animals have to be approved by the Animal Care and Use Committee of the users&#8217; institution, which complies with the Health Guide for the Care and Use of Laboratory Animals of the users&#8217; country.
	<ol>
		<li>Perform mouse euthanasia with the inhalation of 99% CO2 (7 psi) in a Plexiglas chamber (Figure 6A).</li>
		<li>Open mouse abdomen and cut the thoracic aorta to bleed the mouse.</li>
		<li>Remove the diaphragm, the thoracic cage and the lung lobes (Figure 6B). NOTE: To minimize the risk of damaging the tissue, keep the heart attached to the aorta and avoid touching the vessel directly but manipulate it using the heart.</li>
		<li>Remove the heart, the aortic root and the thoracic aorta by gently cutting between the vessel and the spine. NOTE: Do not induce any elongation in the vessel during the excision to keep the inner structure of the tissue intact (Figure 6C).</li>
		<li>Immediately immerse and keep the heart and aorta in Krebs solution.</li>
		<li>Cut and carefully wash the aorta in Krebs solution to remove any blood clots. Remove connective tissue using micro-scissors, tweezers and surgical dissecting microscope (Figure 6D-E). NOTE: Keep all the vessel length and use the aortic root to determine the vessel orientation.</li>
	</ol>
	</li>
	<li>Vessel Dimension Determination and Mounting 
	To determine the stiffness of the vessel, the unloaded vessel dimensions are required and can be determined with a calibrated microscope.
	<ol>
		<li>Cut an aortic ring of about 2 mm in length and precisely measure its length using a calibrated microscope setting (Figures 6F-G). Put this segment aside in Krebs solution.</li>
		<li>Cut another aortic ring as small as possible between each of the 2 mm segments (Figure 6F). Put this small segment on a microscope glass slide with the lumen facing up and measure the wall thickness using a calibrated microscope setting (Figure 6H).</li>
		<li>Fill the Petri dish on the BAXS platform with Krebs solution and insert the 2 mm aorta ring segment on the pulling pins (inset in Figure 4C).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig5highres.jpg" width="500" /> 
Figure 5.&#160;PDMS substrate fabrication and mounting. A) After curing, the substrate is carefully peeled off the SU-8 2050 mold and put aside in a Petri dish. B) Anchoring features made out of PDMS and help to secure the substrate on the clamps. C) Substrate with anchoring features ready for mounting. D) The substrate is mounted on the 4&#160;clamps, which are then mounted on the clamp holder (see inset). E) Detailed picture of the substrate mounted on the 4&#160;clamps. F) Procedure of pouring PDMS in the groove underneath the substrate. The arrow shows the PDMS slowly filling the groove by capillarity.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51454/51454fig6highres.jpg" width="500" /> 
Figure 6.&#160;Preparation and isolation of the thoracic aorta. A) Preparing surgical instruments and the euthanized mouse. B) Through a longitudinal abdominal incision, the thoracic cage and lung lobs are removed. C) The aorta is carefully removed using the heart to manipulate the tissue. D) The heart and aorta are put in Krebs physiological solution. The aorta is cleaned by removing all connective tissues. E) Detailed picture showing the heart and aorta. F) Aorta segment used for stiffness assessment along with small segments used for thickness measurement. G-H) The precise length (G) and thickness (H) of each vessel segments are evaluated using an inverse microscope and an analysis program.

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The authors declare that they have no competing financial interests.

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 ...

a-novel-stretching-platform-for-applications-cell-tissue

Research

JoVE Journal

Bioengineering

11.7K visualizações.  University of Ottawa.  Apresentamos neste artigo uma novela que se estende plataforma que pode ser usada para investigar as respostas das células individuais a complexa deformação mecânica biaxial anisotrópica e quantificar as propriedades mecânicas dos tecidos biológicos. 

Vídeo: A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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

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

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

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...