Name: Preparation of the Microfluidic Device for Electrodeformation of RBCs
Uploaded: 2023-10-13T14:00:00
Description: Watch this Scientific Journal Video about Preparation of the Microfluidic Device for Electrodeformation of RBCs at JoVE.com

preparation of the microfluidic device for electrodeformation of rbcs

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 Preparation of the Microfluidic Device for Electrodeformation of RBCs at JoVE.com

Preparation of the Microfluidic Device for Electrodeformation of RBCs  

적혈구의 전기변형을 위한 미세유체 소자의 제조

먼저 미세유체 채널 설계를 위해 SU8 마스터 실리콘 웨이퍼를 14cm 플라스틱 페트리 접시 내부에 테이프로 붙이고 질소 가스로 세척합니다. 종이컵에 담긴 폴리디메틸실록산 또는 PDMS 베이스 및 PDMS 경화제의 무게를 적당량 측정합니다. 나무 주걱을 사용하여 혼합물이 흐린 흰색이 될 때까지 두 가지를 섞습니다.PDMS 혼합물을 실리콘 웨이퍼가 들어 있는 플라스틱 페트리 접시에 붓습니다. 그런 다음 페트리 접시를 3방향 마개가 장착된 진공 데시케이터에 넣습니다. 마개의 밸브를 돌려 진공을 데시케이터 챔버에 연결하여 혼합물에서 기포를 제거합니다.채널의 기능에서 모든 기포가 제거되면 페트리 접시를 섭씨 70도의 오븐에 4시간 동안 넣습니다. 페트리 접시가 실온으로 식은 후 커팅 매트 위에 놓습니다. 메스를 사용하여 실리콘 웨이퍼 위의 PDMS 부분을 잘라냅니다.컷아웃 PDMS를 실험실 포장 필름의 두 시트 사이에 놓습니다. 마이크로 채널의 움푹 들어간 부분과 필름 사이의 간격은 미세 유체 채널의 입구와 출구를 찾는 데 도움이 됩니다. 그런 다음 면도날을 사용하여 대형 PDMS에서 개별 채널을 잘라내고 각각의 생검 펀치를 사용하여 채널에 적절한 입구 및 출구 구멍을 만듭니다.채널 쪽이 위를 향하도록 구멍이 뚫린 채널을 깨끗한 유리 슬라이드 위에 놓습니다. 박막 인듐-주석 산화물 또는 ITO 간지 전극이 포함된 유리 기판을 전극이 위를 향하도록 하여 동일한 슬라이드에 놓습니다. 그런 다음 유리 슬라이드를 플라즈마 클리너에 부드럽게 넣습니다.가스 밸브를 닫고 펌프를 켠 후 600-800 밀리토르의 센서 판독 값을 얻을 때까지 2 분 동안 기다리십시오. 그런 다음 전원 스위치를 켜고 30초 동안 기다렸다가 RF 전원 노브를 낮음에서 높음으로 돌리고 1분 동안 기다립니다. 그런 다음 세트를 끄려면 역순으로 따르십시오.플라즈마 클리너의 챔버를 연 직후 PDMS를 들어 올려 채널 쪽이 아래를 향하도록 180도 회전합니다. ITO 기판 상단에 채널을 배치하여 접착 공정을 시작합니다. 핀셋을 사용하여 PDMS의 모서리를 약 3초 동안 부드럽게 누릅니다.프라이밍 매체를 23게이지 바늘이 있는 1밀리리터 주사기에 넣습니다. 기포를 도입하지 않고 매체를 방출하기 전에 바늘을 입구에 똑바로 삽입하여 채널을 천천히 조심스럽게 적십니다. 최소 3분 동안 배양한 후 10마이크로리터 피펫 팁을 사용하여 프라임 배지를 제거합니다.마지막으로, 매체를 채널에 삽입하여 유전영동 매체로 채널을 세 번 세척합니다.

preparation-of-the-microfluidic-device-for-electrodeformation-of-rbcs

Research

JoVE Journal

Bioengineering

Preparation of the Microfluidic Device for Electrodeformation of RBCs

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

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.

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

Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC

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.