Name: Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC
Uploaded: 2023-10-13T14:00:00
Description: Watch this Scientific Journal Video about Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC at JoVE.com

preparing the electrodeformation setup and performing fatigue testing on human rbc

진폭 변조 전기변형에 의한 적혈구의 기계적 피로 시험

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.

저자 스포트라이트: 전기변형 거동을 조절하여 순환 세포의 생체 역학 연구 

생물학적 세포의 기계적 피로를 평가하기 위한 진폭 변조 전형성

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 Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC at JoVE.com

Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC  

전극 설정 준비 및 인체 적혈구에 대한 피로 시험 수행

시작하려면 미세 유체 장치를 테스트 픽스처의 바닥 부분에 놓습니다. 고정 장치의 상단 부분을 장치에 맞추고 두 세트의 나일론 나사와 너트를 사용하여 두 부분을 조립합니다. 테스트 픽스처를 현미경 스테이지에 놓습니다.현미경 아래에서 원하는 전극 한 세트를 찾습니다. 설정된 위치의 전극과 일치하는 해당 쌍의 전극 와이어를 함수 발생기의 출력 단자에 연결합니다. 미세유체 채널의 3mm 입구에서 5마이크로리터의 유전영동 또는 DEP 배지를 제거합니다.10마이크로리터 피펫 팁을 사용하여 준비된 적혈구 또는 RBC 현탁액 5마이크로리터를 입구에 천천히 로드하고 세포가 1분 동안 가라앉도록 합니다. 20X 배율에서 채널을 관찰하십시오. 사인 버튼을 누르고 3메가헤르츠의 주파수에서 2볼트 평균 제곱근의 진폭을 갖는 사인파를 정의합니다.Mod 버튼을 눌러 변조를 활성화합니다. Type 옵션을 눌러 웨이브 모드를 진폭 이동 키잉 또는 ASK로 변경합니다. 변조 주파수를 250 메가헤르츠로 설정하며, 이는 4초의 로딩/언로딩 기간에 해당합니다.함수 생성기의 출력을 켜고 초당 30프레임으로 10분마다 1분짜리 비디오를 녹화합니다. 적혈구는 전계 강도가 더 높은 전극의 가장자리로 이동하여 전기 여기(excitation)에 자발적으로 반응했습니다. on-keying 단계에서는 전극 변형으로 인해 적혈구가 늘어났고, off-keying 단계에서는 적혈구가 이완되었습니다.1시간 피로 테스트 동안 개별 적혈구를 추적했을 때 세포 변형의 점진적인 감소가 관찰되었습니다.

preparing-electrodeformation-setup-performing-fatigue-testing-on

Research

JoVE Journal

Bioengineering

Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC

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.

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.

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

연구

생체공학

Preparation of Dielectrophoresis Medium and Electrodeformation Prime Medium

To prepare the dielectrophoresis, or DEP medium, weigh 12.75 grams of sucrose and 0.45 grams of dextrose using a scale. Dissolve both powders in a solution of 150 milliliters of deionized water and 3.5 milliliters of PBS. Using a low-range conductivity tester, measure the conductivity to ensure it is 0.04 siemens per meter.Using an osmometer, confirm that the osmolarity is within the normal range of the blood plasma. Store the prepared DEP medium at four degrees Celsius. In a 15-milliliter tube, dissolve 0.5 grams of bovine serum albumin in 10 milliliters of the DEP medium, and mix thoroughly using a vortex mixer to prepare the device's prime medium.

Dielectrophoresis 매질 및 Electrodeformation Prime Medium의 제조

Preparation of the Microfluidic Device for Electrodeformation of RBCs

To begin, tape the SU8 master silicon wafer for the microfluidic channel design on the inside of a plastic 14-centimeter Petri dish and clean it with nitrogen gas. Weigh appropriate amounts of polydimethylsiloxane, or PDMS base and PDMS curing agent, in a paper cup. Using a wooden spatula, mix the two until the mixture becomes cloudy white in color.Pour the PDMS mixture into the plastic Petri dish containing the silicon wafer. Then, place the Petri dish into a vacuum desiccator equipped with a three-way stopcock. Turn the valve of the stopcock to connect the vacuum to the desiccator chamber to remove air bubbles from the mixture.Once all air bubbles are removed from the features of the channels, place the Petri dish inside an oven at 70 degrees Celsius for four hours. After the Petri dish cools to room temperature, place it on a cutting mat. Using a scalpel, cut out the portion of the PDMS above the silicon wafer.Place the cut-out PDMS between two sheets of the lab wrapping film. The gap between the indent of the microchannel and the film helps to locate the inlet and outlet of the microfluidic channel. Next, using a razor blade, cut out an individual channel from the large PDMS and, with respective biopsy punches, make appropriate inlet and outlet holes in the channel.Place the hole-punched channel with the channel side facing up onto a clean glass slide. Place a glass substrate containing thin film indium-tin oxide, or ITO interdigitated electrodes, on the same slide with the electrodes facing up. Then, gently place the glass slide into a plasma cleaner.After closing the gas valve and switching on the pump, wait for two minutes to obtain a sensor reading of 600 to 800 millitorr. Next, turn on the power switch and wait 30 seconds before turning the RF power knob from low to high and waiting for one minute. Then, to switch off the set, follow the reverse sequence.Immediately after opening the chamber of the plasma cleaner, lift and rotate the PDMS 180 degrees so that the channel side is facing down. Place the channel on the top of the ITO substrate to initiate the bonding process. Using tweezers, gently press down on the corners of the PDMS for about three seconds.Load the priming medium into a one-milliliter syringe with a 23-gauge needle. Slowly and carefully wet the channel by inserting the needle straight into the inlet well before releasing the medium without introducing air bubbles. After incubating for at least three minutes, remove the prime medium using a 10-microliter pipette tip.Finally, wash the channel with dielectrophoresis medium three times by inserting the medium into the channel.

적혈구의 전기변형을 위한 미세유체 소자의 제조

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.

전극 변형 실험을 위한 인간 적혈구 현탁액의 제조

To begin, place the microfluidic device into the bottom part of the test fixture. Align the top part of the fixture onto the device, and using two sets of nylon screws and nuts, assemble the two parts. Place the test fixture on the microscope stage.Locate one desired set of electrodes under the microscope. Connect the corresponding pair of electrode wires that match the located electrode set to the output terminal of the function generator. Remove five microliters of the dielectrophoresis, or DEP medium, from the three-millimeter inlet of the microfluidic channel.Using a 10-microliter pipette tip, slowly load five microliters of the prepared red blood cell, or RBC suspension, into the inlet and allow the cells to settle for one minute. Observe the channel under 20X magnification. Press the Sine button and define a sine wave with an amplitude of two volts root mean square at a frequency of three megahertz.Press the Mod button to enable modulation. Press the Type option to change the wave mode into amplitude shift keying, or ASK. Set the modulation frequency to 250 megahertz, corresponding to a four-second loading/unloading period.Turn on the Output of the function generator and record a one-minute video every 10 minutes at 30 frames per second. The RBCs spontaneously responded to the electrical excitation by moving towards edges of electrodes with higher field strength. Under the on-keying phase, RBCs were stretched due to electrode deformation, while under the off-keying phase, RBCs were relaxed.When tracking individual RBCs during the one-hour fatigue testing, a gradual decrease in cellular deformation was observed.