This research has been funded by NSF/CMMI Mechanobiology of Hemoglobin-Based Artificial Oxygen Carriers (#1941655) and NSF/CMMI Dynamic and Fatigue Analysis of Healthy and Diseased Red Blood Cells (#1635312).

Deidentified human whole blood was commercially obtained. Work involving the blood samples was performed in a biosafety level 2 laboratory utilizing protocols approved by the Institutional Biosafety Committee at Florida Atlantic University.
1. Microfluidic device preparation
<ol>
	<li>Tape down the SU-8 master silicon wafer for the microfluidic channel design on the inside of a plastic 14 cm Petri dish and clean it with N2 gas.</li>
	<li>Weigh 60 g of polydimethylsiloxane (PDMS) base and 6 g of PDMS curing agent in a paper cup. Mix the two parts using a wooden spatula until the mixture is a cloudy white color.</li>
	<li>Pour the PDMS mixture into the plastic Petri dish containing the silicon wafer. Place the Petri dish into a vacuum desiccator with a 3-way stopcock. Turn the valve of the stopcock to connect the vacuum to the desiccator chamber to remove air bubbles from the PDMS.</li>
	<li>Reintroduce air into the desiccator chamber by adjusting the stopcock valve to connect the desiccator chamber to the ambient in approximately 5 min cycles. Repeat until all air bubbles are removed from the features of the channels.</li>
	<li>Place the Petri dish inside an oven at 70 &#730;C for 4 h. Once time has elapsed, remove the Petri dish, allow it to cool down to room temperature, and place it on a cutting mat.</li>
	<li>Using a scalpel, cut out the portion of the PDMS above the silicon wafer. Place the cutout PDMS in between the two sheets of lab-wrapping film. The gap formed between the indent of the microchannel and the semi-transparent film facilitates the identification of the location of the microfluidic channel as well as its respective inlet and outlet.</li>
	<li>Using a razor blade, cut out an individual channel from the large PDMS. Punch a 3 mm inlet hole and 1.5 mm outlet hole using biopsy punches respective to the two sizes (Figure 1A).</li>
	<li>Place the hole-punched channel, with the channel side facing up, onto a clean glass slide. Place a 20 mm x 15 mm glass substrate containing thin-film Indium Tin Oxide (ITO) interdigitated electrodes on the same glass slide with electrodes facing up.</li>
	<li>Gently place the glass slide with PDMS and substrate into a plasma cleaner. Close the gas valve, turn ON the pump switch, and wait 2 min to obtain a sensor reading of 600 - 800 mTorr.</li>
	<li>Turn ON the power switch and wait for 30 s. Turn the RF power knob from low to high and wait for 1 min.</li>
	<li>Then, reverse the sequence by turning the RF knob to the low, power switch to OFF, pump switch to OFF, and opening the gas valve.</li>
	<li>Immediately after opening the chamber of the plasma cleaner, lift and rotate the PDMS so that the channel side is facing down (180&#730;). Place the channel on the top of the ITO substrate. The bonding process has begun.</li>
	<li>Using tweezers, gently press down on the corners of the PDMS for about 3 s. Avoid pressing onto the channel itself. 
	NOTE: The bonding process happens spontaneously upon physical contact between the two treated surfaces.</li>
	<li>Load the priming medium into a 1 mL syringe with a 23 G needle. Carefully wet the channel by inserting the needle straight into the inlet well and then releasing the medium. Operate slowly. Do not introduce air bubbles. Incubate for at least 3 min.</li>
	<li>Remove the prime medium using a 10 &#181;L pipette tip. Wash the channel with DEP medium 3 times by inserting the DEP medium into the channel. Keep the channel wet at all times.</li>
</ol>
2. Test fixture
NOTE: The test fixture is designed using 3D CAD software and includes a base housing unit and a top unit (Figure 1B). Then, it is manufactured using a 3-axis CNC milling machine with a standard tolerance limit of around &#177; 0.005-inch dimension of the test fixture is checked using an electronic caliper (not shown). Sterility of the fixture is not required for the in vitro biomechanical testing.
<ol>
	<li>Pre-solder wires into the solder cup ends of two sets of spring piston connectors.</li>
	<li>Insert the spring piston connectors into the top unit and create a permanent bonding by adding a drop of epoxy glue.</li>
</ol>
3. Preparation of electrodeformation working buffer
<ol>
	<li>To prepare the DEP medium, weigh 12.75 g of sucrose and 0.45 g of dextrose using a scale.</li>
	<li>Dissolve both powders into a single container with 150 mL of deionized (DI) water and 3.5 mL of phosphate-buffered saline (PBS).</li>
	<li>Using a low-range conductivity tester, measure the conductivity and ensure it is 0.04 S/m (Figure 2). 
	NOTE: A different conductivity value can be used, which could change the sign and magnitude of the resultant DEP force11. Electrodeformation, however, requires a positive DEP force.</li>
	<li>Using an osmometer, confirm the osmolarity to be within the normal range of the blood plasma, 275 to 295 mOsm/kg of water (Figure 3). Store at 4 &#730;C. The DEP medium is now prepared.</li>
	<li>In a 15 mL tube, dissolve 0.5 g of bovine serum albumin (BSA) in 10 mL of the DEP medium. Mix thoroughly. The device&#39;s prime medium is now prepared.</li>
</ol>
4. Preparation of cell suspension
<ol>
	<li>Wash 20 &#181;L of the whole blood by centrifuging the blood with 1 mL of PBS at 268 x g for 3 min. Discard the supernatant.</li>
	<li>Resuspend the RBCs in 1 mL of PBS. Gently pipette to mix. Wash the RBCs for 3 min at 268 x g and discard the supernatant.</li>
	<li>Extract 5 &#181;L of RBC pellet using a 10 &#181;L micropipette tip and fully dispense into 1 mL of the DEP medium. Wash the cells by centrifuging at 268 x g for 3 min.</li>
	<li>Discard the supernatant and resuspend the RBCs in 1 mL of DEP medium. Gently pipette to mix.</li>
	<li>Wash the RBCs for 3 min at 268 x g and discard the supernatant. Pipette 2 &#181;L of RBC pellet into 500 &#181;L of DEP medium. The cell suspension is now prepared with a concentration within a range of 1 - 6.2 x 104 cells/&#181;L12, which can be confirmed using a standard cell counting slide.</li>
</ol>
5. Electrodeformation setup and fatigue testing
<ol>
	<li>Place the microfluidic device into the bottom part of the test fixture. Align the top part of the fixture to the device and assemble the two parts using two sets of nylon screws and nuts (Figure 4).</li>
	<li>Place the test fixture on the microscope stage. Locate one desired set of electrodes under the microscope.</li>
	<li>Connect the corresponding pair of electrode wires that match the located electrode set to the output terminal of the function generator (Figure 4).</li>
	<li>Remove 5 &#181;L of the DEP medium from the 3 mm inlet of the microfluidic channel. Slowly load 5 &#181;L of the cell suspension into the inlet using a 10 &#181;L pipette tip.</li>
	<li>Allow cells to settle down for 1 min. If necessary, add an additional DEP medium to the inlet to push cells into the channel.</li>
	<li>Observe the channel under 20x magnification. Use a 414/46 nm bandpass filter to enhance the contrast of the imaging.</li>
	<li>Press the Sine button and define a sine wave with a 2 VRMS amplitude at 3 MHz frequency. Press the Mod button to enable modulation. Change the wave mode into ASK by pressing the Type option.</li>
	<li>Set the modulation frequency to 250 mHz, which corresponds to a 4 s loading-unloading period (Figure 5A). Turn ON the output of the function generator.</li>
	<li>Record a 1 min video every 10 min at 30 frames per s (fps).</li>
</ol>
6. Characterization of RBC deformation
<ol>
	<li>Using a video editing application, open .avi files recorded in the previous step by pressing Ctrl+O. Use the timeline to choose a frame of interest and set the selection start and end frames to be identical by pressing the Home key and then the End key on the keyboard.</li>
	<li>Export the image frame. Select the output format to be JPEG and press OK.</li>
	<li>Open the ImageJ application and load the images saved in the previous step. Begin by setting the needed measurements by pressing Analyze &#62; Set Measurements and ensuring the check boxes for Area, Perimeter, and Fit Ellipse are checked. Press OK.</li>
	<li>Next, convert the image to grayscale by choosing Image &#62; Type &#62; 8-bit.</li>
	<li>Then convert the image to binary using Image &#62; Adjust &#62; Thresholding. In the Threshold dialog box, adjust the two sliders as needed. Press Apply and then close the Threshold dialog box.</li>
	<li>Choose Analyze &#62; Tools &#62; ROI Manager. In the ROI Manager, press the checkbox labeled Show All. Do not close this box.</li>
	<li>Select the Wand (tracing) Tool, select an applicable cell in the image, and press T on the keyboard. The selected cell will be numbered. A new cell can be selected again. Select all applicable cells to be measured. The applicable cells are identified as those that are isolated from other cells. The number of these cells could range from 50 to 200 in a single field of view.</li>
	<li>Return to the ROI Manager box and press Measure. This opens the Results box. Columns labeled Major and Minor are the lengths of the fitted ellipse major and minor axes (in pixels), respectively. Choose File &#62; Save As to export the measurements as a CSV formatted file.</li>
	<li>Using any appropriate computational analysis software, calculate the quotient of Major and Minor.</li>
</ol>

Mechanical Fatigue Testing of RBCs by Amplitude-Modulated Electrodeformation

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+red+cell+lifespan+and+aging.">Measurement of red cell lifespan and aging.</a> Transfusion Medicine and Hemotherapy. 39 (5), 302-307 (2012).</li><li>Matthews, K., Lamoureux, E. S., Myrand-Lapierre, M. -. E., Duffy, S. P., Ma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+for+measuring+red+blood+cell+deformability.">Technologies for measuring red blood cell deformability.</a> Lab on a Chip. 22, 1254-1274 (2022).</li><li>Kim, J., Lee, H., Shin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+measurement+of+red+blood+cell+deformability:+A+brief+review.">Advances in the measurement of red blood cell deformability: A brief review.</a> Journal of Cellular Biotechnology. 1 (1), 63-79 (2015).</li><li>Varga, A., Matrai, A. A., Barath, B., Deak, A., Horvath, L., Nemeth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+diversity+of+osmotic+gradient+deformability+of+red+blood+cells+in+human+and+seven+vertebrate+animal+species.">Interspecies diversity of osmotic gradient deformability of red blood cells in human and seven vertebrate animal species.</a> Cells. 11 (8), 1351 (2022).</li><li>Doh, I., Lee, W. C., Cho, Y. -. H., Pisano, A. P., Kuypers, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformation+measurement+of+individual+cells+in+large+populations+using+a+single-cell+microchamber+array+chip.">Deformation measurement of individual cells in large populations using a single-cell microchamber array chip.</a> Applied Physics Letters. 100 (17), 173702 (2012).</li><li>Al Safi, A., Bazuin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+digital+transmitters+with+amplitude+shift+keying+and+quadrature+amplitude+modulators+implementation+examples.">Toward digital transmitters with amplitude shift keying and quadrature amplitude modulators implementation examples.</a> , 1-7 (2017).</li><li>Zhang, J., Chen, K., Fan, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cell+isolation+and+analysis.">Circulating tumor cell isolation and analysis.</a> Advances in Clinical Chemistry. 75, 1-31 (2016).</li><li>Cottet, J., Fabregue, O., Berger, C., Buret, F., Renaud, P., Fr&#233;n&#233;a-Robin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyDEP:+a+new+computational+tool+for+dielectric+modeling+of+particles+and+cells.">MyDEP: a new computational tool for dielectric modeling of particles and cells.</a> Biophysical Journal. 116 (1), 12-18 (2019).</li><li>Haywood, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+the+full+blood+count.">Interpreting the full blood count.</a> InnovAiT. 15 (3), 131-137 (2022).</li><li>Qiang, Y., Liu, J., Dao, M., Suresh, S., Du, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+fatigue+of+human+red+blood+cells.">Mechanical fatigue of human red blood cells.</a> Proceedings of the National Academy of Sciences. 116 (40), 19828-19834 (2019).</li><li>Gharaibeh, B., 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=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nature Protocols. 3 (9), 1501-1509 (2008).</li><li>Qiang, Y., Liu, J., Dao, M., Du, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assay+for+single-cell+characterization+of+impaired+deformability+in+red+blood+cells+under+recurrent+episodes+of+hypoxia.">In vitro assay for single-cell characterization of impaired deformability in red blood cells under recurrent episodes of hypoxia.</a> Lab on a Chip. 21 (18), 3458-3470 (2021).</li></ol>

The authors have nothing to disclose.

The ASK OOK modulation of a DEP force-inducing sine wave can be used to test the mechanical fatigue of RBCs over a long period of time. In this protocol, we limited the in vitro fatigue testing to 1 hour to prevent the potential adverse metabolic effects on the cell deformability. Comprehensive fatigue testing conditions can be programmed using the ASK-modulated electrodeformation technique. Parameters such as loading frequency, amplitude, and loading rate can all be programmed. The loading frequency can be programmed to...

Red blood cells (RBCs) are the most deformable cells in the human body1. Their deformability is directly related to their oxygen-carrying functionality. Reduced deformability in RBCs has been found to correlate with the pathogenesis of several RBC disorders2. Deformability measurements have led us to a better understanding of RBC-related diseases3. The normal lifespan of RBCs can vary from 70 to 140 day4. Therefore, it is important to measure how their deformability decreases along with the aging process, e.g., their fatigue behavior due to cyclic shear stresses3.
Measuring RBC deformability at high throughput is challenging because of the piconewton scale forces (~10-12 N) that are applied to the individual cells. Over the past decade, many technologies have been developed to measure cell deformability5. Deformation measurements of RBCs at the single-cell level can be performed by pipette aspiration and optical tweezers, while bulk analyses are done by osmotic gradient ektacytometry. Ektacytometry analyses provide an abundance of data, which provides an opportunity to diagnose blood disorders6,7. The deformability of RBCs can also be analyzed using the viscoelastic theory by colloid probe atomic force microscopy. In this method, computational analysis is applied to estimate the elastic modulus of RBCs, considering both time-dependent and steady-state responses. The deformability of individual RBCs can be measured by using the single-cell microchamber array method. This method analyzes each cell through the membrane and cytosolic fluorescent markers to provide information for RBC deformability and the distribution of cellular characteristics in complex RBC populations to detect hematologic disorders8.
Fatigue is a key factor in the degradation of properties of engineered materials and biomaterials. Fatigue testing enables a quantitative analysis of the integrity and longevity of a structure subjected to cyclic loading. Analysis of fatigue in biological cells has long been hampered by the lack of a general, readily applicable, high throughput, and quantitative method for the implementation of cyclic deformation in cell membranes. This is possible with the utilization of electrical signal modulation and electrodeformation techniques implemented in a microfluidic setting. The amplitude shift keying (ASK) technique as a digital modulation is applied through On-Off keying (OOK) modulation in this article. The concept of keying refers to the transmission of digital signals over the channel, which requires a sine wave carrier signal to function9. The ON and OFF times can be set equal. Under ON-keying, RBCs enter a deformed state while exposed to an external electrodeformation force (Fdep)10 created by the nonuniform electric field. Under OFF-keying, RBCs are in their relaxed state. We observe the fatigue of RBCs, namely a progressive degradation in their ability to stretch with increasing loading cycles. The fatigue-induced deformability loss in RBCs can provide insights into the accumulated membrane damage during blood circulation, enabling us to further investigate the connections between cell fatigue and disease states.
Here we provide step-by-step procedures on how fatigue testing of RBCs is implemented in a microfluidic device via ASK-modulated electrodeformation and the system settings such as microfluidic device, mechanical loading, and microscopic imagining for the characterization of the gradual degradation in mechanical deformability of RBCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Balance Scale</td>
			<td>ViBRA</td>
			<td>HT-224R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>BRIGHTLINE</td>
			<td>414/46 BrightLine HC</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Disposable Syringes with Luer-Lok&#8482; Tips, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punches with Plunger System, 1.5 mm</td>
			<td>Fisher Scientific</td>
			<td>12-460-403</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punches with Plunger System, 3 mm</td>
			<td>Fisher Scientific</td>
			<td>12-460-407</td>
			<td>1.5 mm and 3 mm diameter</td>
		</tr>
		<tr>
			<td>Blunt needle, 23-gauge</td>
			<td>BSTEAN</td>
			<td>X001308N97</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin</td>
			<td>RMBIO</td>
			<td>BSA-BSH</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SCILOGEX</td>
			<td>911015119999</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tube, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>MDX01455</td>
			<td>MilliporeSigma&#8482;</td>
		</tr>
		<tr>
			<td>EC Low Conductivity meter</td>
			<td>ecoTestr</td>
			<td>358/03</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160; &#160;Snap-Cap MicrocentrifugeTubes</td>
			<td>www.eppendorf.com</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft&#160;</td>
			<td></td>
			<td>Graph plotting</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>SIGLENT</td>
			<td>SDG830</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass/ITO Electrode Substrate</td>
			<td>OSSILA</td>
			<td>S161</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>IX81 - SN9E07015</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Oven</td>
			<td>QUINCY LAB (QL)</td>
			<td>MODEL 30GCE</td>
			<td>Digital Model</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>Graph plotting</td>
		</tr>
		<tr>
			<td>Micro Osmometer - Model 3300</td>
			<td>Advanced Instruments Inc.</td>
			<td>S/N: 03050397P</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm Laboratory Wrapping Film</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>FALCON</td>
			<td>SKU=351006</td>
			<td>ICSI/Biopsydish 50*9 mm</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>LONZA</td>
			<td>04-479Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick plasma PDCOOL</td>
			<td>NC0301989</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td>Dassault Systemes</td>
			<td></td>
			<td>CAD software</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>50-188-2419</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>SPBEL-ART</td>
			<td>F42400-2121</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden spatula</td>
			<td>Fisher Scientific</td>
			<td>NC0304136</td>
			<td>Tongue Depressors Wood NS 6&#34;</td>
		</tr>
	</tbody>
</table>

amplitude-modulated electrodeformation to evaluate mechanical fatigue of biological cells

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the microcirculation. Reduced deformability is seen in physiologically aged RBCs. Existing techniques to measure cell deformability cannot easily be used for measuring fatigue, the gradual degradation in cell membranes caused by cyclic loads. We present a protocol to evaluate mechanical degradation in RBCs from cyclic shear stresses using amplitude shift keying (ASK) modulation-based electrodeformation in a microfluidic channel. Briefly, the interdigitated electrodes in the microfluidic channel are excited with a low voltage alternating current at radio frequencies using a signal generator. RBCs in suspension respond to the electric field and exhibit positive dielectrophoresis (DEP), which moves cells to the electrode edges. Cells are then stretched due to the electrical forces exerted on the two cell halves, resulting in uniaxial stretching, known as electrodeformation. The level of shear stress and the resultant deformation can be easily adjusted by changing the amplitude of the excitation wave. This enables quantifications of nonlinear deformability of RBCs in response to small and large deformations at high throughput. Modifying the excitation wave with the ASK strategy induces cyclic electrodeformation with programmable loading rates and frequencies. This provides a convenient way for the characterization of RBC fatigue. Our ASK-modulated electrodeformation approach enables, for the first time, a direct measurement of RBC fatigue from cyclic loads. It can be used as a tool for general biomechanical testing, for analyses of cell deformability and fatigue in other cell types and diseased conditions, and can also be combined with strategies to control the microenvironment of cells, such as oxygen tension and biological and chemical cues.

When cell suspension was loaded in the microfluidic channel, a relatively uniform distribution of cells was observed. Upon the signal output (e.g., a simple sine wave or On-Keying phase of ASK) from the function generator, the thin-film interdigitated electrodes generated a nonuniform alternating current electrical field. The suspended cells spontaneously responded to this electrical excitation and exhibited a positive DEP behavior, namely moving towards the edges of electrodes with higher field strength. Consequently, c...

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Author Spotlight: Studying Biomechanics of Circulating Cells by Modulating Their Electrodeformation Behavior 

Presented here is a protocol for mechanical fatigue testing in the case of human red blood cells using an amplitude-modulated electrodeformation approach. This general approach can be used to measure the systematic changes in morphological and biomechanical characteristics of biological cells in a suspension from cyclic deformation.

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Red blood cells (RBCs) are known for their remarkable deformability. They repeatedly undergo considerable deformation when passing through the ...

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1.4K Views.  Florida Atlantic University. Presented here is a protocol for mechanical fatigue testing in the case of human red blood cells using an amplitude-modulated electrodeformation approach. This general approach can be used to measure the systematic changes in morphological and biomechanical characteristics of biological cells in a suspension from cyclic deformation. 

Video: Author Spotlight: Studying Biomechanics of Circulating Cells by Modulating Their Electrodeformation Behavior 

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
	Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. 
	We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...