Name: Preparation of Human RBC Suspension for Electrodeformation Experiment
Uploaded: 2023-10-13T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Human RBC Suspension for Electrodeformation Experiment at JoVE.com

preparation of human rbc suspension for electrodeformation experiment

Mechanical Fatigue Testing of RBCs by Amplitude-Modulated Electrodeformation

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.

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.

Watch this Scientific Journal Video about Preparation of Human RBC Suspension for Electrodeformation Experiment at JoVE.com

Preparation of Human RBC Suspension for Electrodeformation Experiment  

To begin wash 20 microliters of the whole blood by centrifuging it with one milliliter of PBS at 268 G for three minutes. Then discard the supernatant. Resuspend the resultant red blood cells, or RBCs, in one milliliter of PBS by gentle pipetting.Wash the RBCs again and discard the supernatant as demonstrated previously. Using a 10-microliter micro-pipette tip, extract five microliters of RBC pellet and fully dispense it into one milliliter of the dielectrophoresis, or DEP, medium. Wash the cells and discard the supernatant as demonstrated.Resuspend the pelleted RBCs in one milliliter of DEP medium by gentle pipetting. After washing the cells and discarding the supernatant, pipette two microliters of RBC pellet into 500 microliters of DEP medium. Confirmed the concentration of the resulting cell suspension using a standard cell counting slide.