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

通过调幅电变形对 RBC 进行机械疲劳测试

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.

作者聚焦：通过调节循环细胞的电变形行为来研究循环细胞的生物力学 

调幅电形成评估生物细胞的机械疲劳

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  

红细胞电变形微流控装置的制备

首先，将用于微流体通道设计的 SU8 主硅晶圆贴在 14 厘米塑料培养皿的内部，并用氮气清洁。在纸杯中称取适量的聚二甲基硅氧烷或PDMS碱和PDMS固化剂。用木铲将两者混合，直到混合物变成浑浊的白色。将PDMS混合物倒入含有硅晶片的塑料培养皿中。然后，将培养皿放入配备有三通旋塞阀的真空干燥器中。转动旋塞阀的阀门，将真空连接到干燥室，以去除混合物中的气泡。从通道特征中去除所有气泡后，将培养皿放入 70 摄氏度的烤箱中四个小时。培养皿冷却至室温后，将其放在切割垫上。使用手术刀，切出硅晶圆上方的PDMS部分。将切出的 PDMS 放在两张实验室包装膜之间。微通道的凹痕与薄膜之间的间隙有助于定位微流控通道的入口和出口。接下来，使用剃须刀片，从大型PDMS上切出一个单独的通道，并使用相应的活检冲头，在通道中打出适当的入口和出口孔。将打孔的通道，通道面朝上放在干净的载玻片上。将含有薄膜铟锡氧化物或ITO叉间电极的玻璃基板放在电极朝上的同一载玻片上。然后，轻轻地将载玻片放入等离子清洗器中。关闭燃气阀并打开泵后，等待两分钟以获得 600 至 800 毫托的传感器读数。接下来，打开电源开关并等待 30 秒，然后将射频功率旋钮从低电平转到高电平并等待一分钟。然后，要关闭该装置，请按照相反的顺序操作。打开等离子清洗器腔室后，立即将 PDMS 抬起并旋转 180 度，使通道侧朝下。将通道放在 ITO 基板的顶部以启动粘合过程。使用镊子，轻轻按压 PDMS 的角约三秒钟。用 23 号针头将启动介质装入一毫升注射器中。在释放介质之前，将针头直接插入入口孔中，缓慢而小心地润湿通道，而不会引入气泡。孵育至少三分钟后，使用 10 微升移液器吸头取出原培养基。最后，将介质插入通道中，用介电泳介质洗涤通道三次。

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 ...

科研

生物工程

Preparation of Dielectrophoresis Medium and Electrodeformation Prime Medium

介电泳介质和电变形素材的制备

Preparation of Human RBC Suspension for Electrodeformation Experiment

用于电变形实验的人红细胞悬浮液的制备

Preparing the Electrodeformation Setup and Performing Fatigue Testing on Human RBC

准备电变形装置并对人红细胞进行疲劳测试