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

<ol><li>Kim, Y., Kim, K., Park, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+techniques+for+red+blood+cell+deformability%3A+recent+advances%5BTitle%5D)+AND+%22Blood+Cell%E2%80%94An+Overview+of+Studies+in+Hematology%22%5BJournal%5D)">Measurement techniques for red blood cell deformability: recent advances.</a> Blood Cell—An Overview of Studies in Hematology. 10, 167-194 (2012).</li>
<li>Safeukui, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+sensing+and+sequestration+of+spherocytic+erythrocytes+by+the+human+spleen%5BTitle%5D)+AND+%22Blood%2C+The+Journal+of+the+American+Society+of+Hematology%22%5BJournal%5D)">Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen.</a> Blood, The Journal of the American Society of Hematology. 120 (2), 424-430 (2012).</li>
<li>Naghedi-Baghdar, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+diet+on+blood+viscosity+in+healthy+humans%3A+a+systematic+review%5BTitle%5D)+AND+%22Electronic+physician%22%5BJournal%5D)">Effect of diet on blood viscosity in healthy humans: a systematic review.</a> Electronic physician. 10 (3), 6563(2018).</li>
<li>Franco, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+red+cell+lifespan+and+aging%5BTitle%5D)+AND+%22Transfusion+Medicine+and+Hemotherapy%22%5BJournal%5D)">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/pubmed?term=((Technologies+for+measuring+red+blood+cell+deformability%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Advances+in+the+measurement+of+red+blood+cell+deformability%3A+A+brief+review%5BTitle%5D)+AND+%22Journal+of+Cellular+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Interspecies+diversity+of+osmotic+gradient+deformability+of+red+blood+cells+in+human+and+seven+vertebrate+animal+species%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Deformation+measurement+of+individual+cells+in+large+populations+using+a+single-cell+microchamber+array+chip%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">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>Toward digital transmitters with amplitude shift keying and quadrature amplitude modulators implementation examples. Al Safi, A., Bazuin, B. 2017 IEEE 7th Annual Computing and Communication Workshop and Conference (CCWC), , 1-7 (2017).</li>
<li>Zhang, J., Chen, K., Fan, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cell+isolation+and+analysis%5BTitle%5D)+AND+%22Advances+in+Clinical+Chemistry%22%5BJournal%5D)">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., Frénéa-Robin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MyDEP%3A+a+new+computational+tool+for+dielectric+modeling+of+particles+and+cells%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">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/pubmed?term=((Interpreting+the+full+blood+count%5BTitle%5D)+AND+%22InnovAiT%22%5BJournal%5D)">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/pubmed?term=((Mechanical+fatigue+of+human+red+blood+cells%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((In+vitro+assay+for+single-cell+characterization+of+impaired+deformability+in+red+blood+cells+under+recurrent+episodes+of+hypoxia%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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><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&amp;nbsp;Disposable Syringes with Luer-Lok&amp;trade; 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&amp;trade;</td></tr><tr><td>EC Low Conductivity meter</td><td>ecoTestr</td><td>358/03</td><td></td></tr><tr><td>Eppendorf&amp;nbsp; &amp;nbsp;Snap-Cap MicrocentrifugeTubes</td><td>www.eppendorf.com</td><td>05-402-25</td><td></td></tr><tr><td>Excel</td><td>Microsoft&amp;nbsp;</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&quot;</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...

Watch this Scientific Journal Video about Studying Biomechanics of Circulating Cells by Modulating Their Electrodeformation Behavior at JoVE.com

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 ...

amplitude-modulated-electrodeformation-to-evaluate-mechanical-fatigue

Research

Education

JoVE Journal

JoVE Core

Electrical Engineering

Bioengineering

Unit 1

Operational Amplifiers

SCIENTISTS IN ACTION

1.5K Views.  Florida Atlantic University. Ons onderzoek is gericht op de studie van de celbiomechanica van circulerende cellen, zoals menselijke rode bloedcellen. We maken gebruik van elektrokinetiek, microfluïdica en materiaalkunde om de mechanische oorsprong van schade aan celmembranen te begrijpen, evenals de mechanismen die ten grondslag liggen aan de verkorte levensduur van bloedcellen bij bepaalde ziekten. Vermoeidheidsstudie van biologische cellen is een uitdaging.Het vraagt om het toepassen van cyclische belastingen o...

Video: Auteur in de schijnwerpers: biomechanica van circulerende cellen bestuderen door hun elektrovervormingsgedrag te moduleren 

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, ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

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 ...

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

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

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

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

Biology

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Samenvatting

Meer video's verkennen