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 polydimethyl...

Mechanical Fatigue Testing of RBCs by Amplitude-Modulated Electrodeformation

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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.

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			<td>Balance Scale</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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			<td>MilliporeSigma&#8482;</td>
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			<td>ecoTestr</td>
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			<td>Eppendorf&#160; &#160;Snap-Cap MicrocentrifugeTubes</td>
			<td>www.eppendorf.com</td>
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			<td>Microsoft&#160;</td>
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			<td>ImageJ</td>
			<td>NIH</td>
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			<td>https://imagej.nih.gov/ij/</td>
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			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
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			<td>Micro Osmometer - Model 3300</td>
			<td>Advanced Instruments Inc.</td>
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			<td>Parafilm Laboratory Wrapping Film</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
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			<td>Petri dish</td>
			<td>FALCON</td>
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			<td>04-479Q</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
			<td>NC0304136</td>
			<td>Tongue Depressors Wood NS 6&#34;</td>
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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.

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

Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Our research is focused on the study of cell biomechanics of circulating cells, such as human red blood cells. We leverage electrokinetics, microfluidics, and material science to understand the mechanical origins of damage in cell membranes, as well as the mechanisms underlying the shortened lifespan of blood cells in certain diseases. Fatigue study of biological cells is challenging.It request application of cyclic loads to cell membranes and tracking the deformation in individual cells. Using amplitude sheet key for ASK to modulate electrode deformation behavior of red blood cells, we could quantify how cell deformability changes aligned with the loading cycles. We demonstrated for the first time that red blood cells'membranes can be degraded by cyclic stretching alone.Our protocol utilizes dielectrophoresis to move cells to the electrode edges for electrode deformation measurement. It does not require any stabilization technique and can be used for cell suspension directly. Using interdigitated microelectrodes allows us to measure tens of cells in a single field of view.

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

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.