这项研究由NSF / CMMI基于血红蛋白的人造氧气载体的机械生物学（#1941655）和NSF / CMMI健康和患病红细胞的动态和疲劳分析（#1635312）资助。

去识别的人全血是商业获得的。涉及血液样本的工作是在生物安全2级实验室进行的，该实验室利用佛罗里达大西洋大学机构生物安全委员会批准的协议进行。
1. 微流控装置制备
<ol><li>在塑料 14 厘米培养皿内侧用胶带将用于微流体通道设计的 SU-8 主硅晶片用胶带粘住，并用 N2 气体清洁。</li>
	<li>在纸杯中称取60克聚二甲基硅氧烷（PDMS）基料和6克PDMS固化剂。用木刮刀将两部分混合，直到混合物呈白色。</li>
	<li>将PDMS混合物倒入含有硅晶片的塑料培养皿中。将培养皿放入带有3通旋塞阀的真空干燥器中。转动旋塞阀的阀门，将真空连接到干燥器室，以去除PDMS中的气泡。</li>
	<li>通过调整旋塞阀，在大约 5 分钟的周期内将干燥器室连接到环境，将空气重新引入干燥器室。重复上述步骤，直到从通道特征中去除所有气泡。</li>
	<li>将培养皿置于70°C的烤箱中4小时。时间过后，取出培养皿，让它冷却至室温，然后将其放在切割垫上。</li>
	<li>使用手术刀，切掉硅晶圆上方的PDMS部分。将切口PDMS放在两张实验室包装膜之间。微通道的压痕和半透明薄膜之间形成的间隙有助于识别微流体通道的位置及其各自的入口和出口。</li>
	<li>使用剃须刀片，从大型PDMS中切出单个通道。使用两种尺寸的活检打孔打孔3 mm入口孔和1.5 mm出口孔（图1A）。</li>
	<li>将打孔通道放在干净的载玻片上，通道面朝上。将含有薄膜氧化铟锡 （ITO） 指叉电极的 20 mm x 15 mm 玻璃基板放在同一载玻片上，电极朝上。</li>
	<li>轻轻地将带有PDMS和基材的载玻片放入等离子清洁器中。关闭燃气阀，打开泵开关，等待 2 分钟以获得 600 - 800 mTorr 的传感器读数。</li>
	<li>打开电源开关并等待 30 秒。将射频电源旋钮从低到高，等待 1 分钟。</li>
	<li>然后，通过将射频旋钮转到低电平、电源开关变为关闭、泵开关变为关闭并打开燃气阀来反转顺序。</li>
	<li>打开等离子清洗器的腔室后，立即抬起并旋转PDMS，使通道侧朝下（180°）。将通道放在ITO基板的顶部。粘合过程已经开始。</li>
	<li>使用镊子轻轻按下 PDMS 的角落约 3 秒。避免按压通道本身。 注意：粘合过程在两个处理过的表面之间的物理接触时自发发生。</li>
	<li>用23 G针头将灌注培养基装入1 mL注射器中。通过将针头直接插入入口孔然后释放介质来小心润湿通道。缓慢操作。不要引入气泡。孵育至少3分钟。</li>
	<li>使用 10 μL 移液器吸头取出优质培养基。通过将 DEP 培养基插入通道，用 DEP 培养基清洗通道 3 次。始终保持通道湿润。</li>
</ol>2. 测试治具
注意：测试夹具使用3D CAD软件设计，包括一个基础外壳单元和一个顶部单元（图1B）。然后，使用标准公差限制约为±3轴CNC铣床制造，使用电子卡尺（未显示）检查测试夹具的0.005英寸尺寸。体外生物力学测试不需要夹具的无菌性。
<ol><li>将焊丝预焊到两组弹簧活塞连接器的焊杯末端。</li>
	<li>将弹簧活塞连接器插入顶部单元，并通过添加一滴环氧胶水形成永久粘合。</li>
</ol>3.电形成工作缓冲液的制备
<ol><li>要制备DEP培养基，请使用秤称取12.75克蔗糖和0.45克葡萄糖。</li>
	<li>将两种粉末溶解到装有 150 mL 去离子 （DI） 水和 3.5 mL 磷酸盐缓冲盐水 （PBS） 的单个容器中。</li>
	<li>使用低量程电导率测试仪测量电导率并确保其为 0.04 S/m（图 2）。 注意：可以使用不同的电导率值，这可能会改变合计 DEP 力11 的符号和大小。然而，电形成需要正的DEP力。</li>
	<li>使用渗透压计确认渗透压在血浆的正常范围内，即275至295 mOsm / kg水（图3）。储存在4°C。DEP 培养基现已准备就绪。</li>
	<li>在 15 mL 管中，将 0.5 g 牛血清白蛋白 （BSA） 溶解在 10 mL DEP 培养基中。充分混合。该设备的主要培养基现已准备就绪。</li>
</ol>4. 细胞悬液的制备
<ol><li>用 1 mL PBS 以 268 x g 离心血液 3 分钟，洗涤 20 μL 全血。弃去上清液。</li>
	<li>将红细胞重悬于 1 mL PBS 中。轻轻移液混合。以268× g 洗涤红细胞3分钟，弃去上清液。</li>
	<li>使用 10 μL 微量移液器吸头提取 5 μL RBC 沉淀，并完全分配到 1 mL DEP 培养基中。通过以268× g 离心3分钟来洗涤细胞。</li>
	<li>弃去上清液并将红细胞重悬于 1 mL DEP 培养基中。轻轻移液混合。</li>
	<li>以268× g 洗涤红细胞3分钟，弃去上清液。将 2 μL 红细胞沉淀移液到 500 μL DEP 培养基中。现在制备的细胞悬液浓度在62 - 104个细胞/ μL12的范围内，可以使用标准细胞计数玻片进行确认。</li>
</ol>5. 电形成设置和疲劳测试
<ol><li>将微流体装置放入测试夹具的底部。将夹具的顶部与设备对齐，并使用两组尼龙螺钉和螺母组装两个部件（图4）。</li>
	<li>将测试夹具放在显微镜载物台上。在显微镜下找到一组所需的电极。</li>
	<li>将与定位的电极组匹配的相应一对电极线连接到函数发生器的输出端子（图4）。</li>
	<li>从微流体通道的 3 mm 入口处取出 5 μL DEP 培养基。使用 10 μL 移液器吸头将 5 μL 细胞悬液缓慢加载到入口中。</li>
	<li>让细胞稳定1分钟。如有必要，在入口处添加额外的DEP培养基以将细胞推入通道。</li>
	<li>在 20 倍放大镜下观察通道。使用 414/46 nm 带通滤光片来增强成像的对比度。</li>
	<li>按下 正弦 波按钮并定义一个在3 MHz频率下幅度为2 VRMS 的正弦波。按 Mod 按钮启用调制。通过按 类型 选项将波形模式更改为 ASK。</li>
	<li>将调制频率设置为 250 mHz，对应于 4 秒的加载-卸载周期（图 5A）。打开函数发生器的输出。</li>
	<li>以每秒 10 帧 （fps） 的速度每 10 分钟录制一段 30 分钟的视频。</li>
</ol>6. 红细胞变形的表征
<ol><li>使用视频编辑应用程序，按 Ctrl+O 打开上一步中记录.avi文件。使用时间轴选择感兴趣的帧，并通过按键盘上的 Home 键和 End 键将选择开始帧和结束帧设置为相同。</li>
	<li>导出图像框。选择要为 JPEG 的输出格式，然后按 确定。</li>
	<li>打开 ImageJ 应用程序并加载上一步中保存的图像。首先，通过按 分析>设置测量 值来设置所需的测量值，并确保选中 “面积”、“周长 ”和 “拟合椭圆 ”复选框。按 确定。</li>
	<li>接下来，通过选择“图像 >类型”> 8 位将图像转换为灰度。</li>
	<li>然后使用图像 >调整>阈值将图像转换为二进制。在“阈值”对话框中，根据需要调整两个滑块。按“ 应用” ，然后关闭“阈值”对话框。</li>
	<li>选择 “分析>工具”>“ROI 管理器”。在ROI管理器中，按标有 “全部显示”的复选框。不要关闭此框。</li>
	<li>选择 魔杖（描摹）工具，选择图像中适用的单元格，然后按键盘上的 T。所选单元格将被编号。可以再次选择新单元格。选择要测量的所有适用单元格。适用的细胞被标识为与其他细胞分离的细胞。在单个视场中，这些像元的数量可以从 50 到 200 个不等。</li>
	<li>返回到“ROI 管理器”框，然后按 “度量”。这将打开“结果”框。标记为“主要”和“次要”的列分别是拟合椭圆长轴和短轴的长度（以像素为单位）。选择 “文件>另存为 ”，将测量值导出为 CSV 格式的文件。</li>
	<li>使用任何适当的计算分析软件，计算主要和次要的商。</li>
</ol>

通过调幅电变形对 RBC 进行机械疲劳测试

<ol>
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作者没有什么可透露的。

DEP 力诱导正弦波的 ASK OOK 调制可用于长时间测试红细胞的机械疲劳。在该协议中，我们将体外疲劳测试限制在1小时，以防止对细胞变形能力的潜在不良代谢影响。可以使用ASK调制电形成技术对综合疲劳测试条件进行编程。加载频率、幅度和加载速率等参数都可以编程。加载频率可以编程为不同的值，以确定疲劳对加载频率的依赖性以及循环加载和静态加载之间的差异13。
<p c...

红细胞（RBC）是人体中最易变形的细胞1。它们的变形性与其携氧功能直接相关。已发现红细胞变形性降低与几种红细胞疾病的发病机制相关2。变形性测量使我们更好地了解了红细胞相关疾病3。红细胞的正常寿命可以从 70 天到 140天不等4。因此，测量它们的变形性如何随着老化过程而降低非常重要，例如，它们由于循环剪切应力引起的疲劳行为3。
在高通量下测量红细胞变形性具有挑战性，因为皮克顿尺度力（~10-12 N）施加到单个细胞上。在过去的十年中，已经开发了许多技术来测量细胞变形性5。红细胞水平的变形测量可以通过移液管抽吸和光镊进行，而批量分析则通过渗透梯度 ektacyto 仪完成。Ektacytometry分析提供了丰富的数据，这为诊断血液疾病提供了机会6,7。红细胞的变形性也可以通过胶体探针原子力显微镜使用粘弹性理论进行分析。该方法应用计算分析来估计RBC的弹性模量，同时考虑了时变和稳态响应。单个红细胞的变形能力可以通过使用单细胞微室阵列方法进行测量。该方法通过膜和胞质荧光标记物分析每个细胞，为红细胞变形性和复杂红细胞群中细胞特征的分布提供信息，以检测血液系统疾病8。
疲劳是工程材料和生物材料性能退化的关键因素。疲劳测试可以对承受循环载荷的结构的完整性和寿命进行定量分析。长期以来，由于缺乏一种通用的、易于应用的、高通量和定量的方法来实施细胞膜中的循环变形，生物细胞疲劳分析一直受到阻碍。这可以通过在微流体环境中利用电信号调制和电形成技术来实现。本文通过开-关键控（OOK）调制应用幅度移键控（ASK）技术作为数字调制。键控的概念是指通过通道传输数字信号，这需要正弦波载波信号才能正常工作9。开和关时间可以设置为相等。在ON键控下，红细胞在暴露于由非均匀电场产生的外部电形成力（Fdep）10 时进入变形状态。在“脱键”下，RBC 处于放松状态。我们观察到红细胞的疲劳，即随着加载周期的增加，它们的拉伸能力逐渐下降。红细胞中疲劳诱导的变形性丧失可以提供对血液循环过程中累积的膜损伤的见解，使我们能够进一步研究细胞疲劳与疾病状态之间的联系。
在这里，我们提供了有关如何通过ASK调制电形成在微流体装置中实施红细胞疲劳测试的分步程序，以及微流体装置，机械负载和微观想象等系统设置，以表征红细胞机械变形的逐渐退化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Balance Scale</td>
			<td>ViBRA</td>
			<td>HT-224R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>BRIGHTLINE</td>
			<td>414/46 BrightLine HC</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Disposable Syringes with Luer-Lok&#8482; Tips, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punches with Plunger System, 1.5 mm</td>
			<td>Fisher Scientific</td>
			<td>12-460-403</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punches with Plunger System, 3 mm</td>
			<td>Fisher Scientific</td>
			<td>12-460-407</td>
			<td>1.5 mm and 3 mm diameter</td>
		</tr>
		<tr>
			<td>Blunt needle, 23-gauge</td>
			<td>BSTEAN</td>
			<td>X001308N97</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin</td>
			<td>RMBIO</td>
			<td>BSA-BSH</td>
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			<td>Centrifuge</td>
			<td>SCILOGEX</td>
			<td>911015119999</td>
			<td></td>
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		<tr>
			<td>Conical Tube, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>MDX01455</td>
			<td>MilliporeSigma&#8482;</td>
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		<tr>
			<td>EC Low Conductivity meter</td>
			<td>ecoTestr</td>
			<td>358/03</td>
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			<td>Eppendorf&#160; &#160;Snap-Cap MicrocentrifugeTubes</td>
			<td>www.eppendorf.com</td>
			<td>05-402-25</td>
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			<td>Excel</td>
			<td>Microsoft&#160;</td>
			<td></td>
			<td>Graph plotting</td>
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			<td>Function Generator</td>
			<td>SIGLENT</td>
			<td>SDG830</td>
			<td></td>
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			<td>Glass/ITO Electrode Substrate</td>
			<td>OSSILA</td>
			<td>S161</td>
			<td></td>
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			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
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			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>IX81 - SN9E07015</td>
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			<td>Lab Oven</td>
			<td>QUINCY LAB (QL)</td>
			<td>MODEL 30GCE</td>
			<td>Digital Model</td>
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			<td>Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>Graph plotting</td>
		</tr>
		<tr>
			<td>Micro Osmometer - Model 3300</td>
			<td>Advanced Instruments Inc.</td>
			<td>S/N: 03050397P</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm Laboratory Wrapping Film</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>FALCON</td>
			<td>SKU=351006</td>
			<td>ICSI/Biopsydish 50*9 mm</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>LONZA</td>
			<td>04-479Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick plasma PDCOOL</td>
			<td>NC0301989</td>
			<td></td>
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		<tr>
			<td>Solidworks</td>
			<td>Dassault Systemes</td>
			<td></td>
			<td>CAD software</td>
		</tr>
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			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>50-188-2419</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>SPBEL-ART</td>
			<td>F42400-2121</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden spatula</td>
			<td>Fisher Scientific</td>
			<td>NC0304136</td>
			<td>Tongue Depressors Wood NS 6&#34;</td>
		</tr>
	</tbody>
</table>

amplitude-modulated electrodeformation to evaluate mechanical fatigue of biological cells

红细胞（RBC）以其显着的变形性而闻名。它们在通过微循环时反复发生相当大的变形。在生理老化的红细胞中可见变形性降低。 现有的测量细胞变形性的技术不容易用于测量疲劳，即由循环负荷引起的细胞膜逐渐退化。我们提出了一种协议，用于在微流体通道中使用基于振幅偏移键控（ASK）调制的电形成来评估循环剪切应力对红细胞的机械降解。简而言之，微流体通道中的叉指电极使用信号发生器以射频的低压交流电激励。悬浮液中的红细胞响应电场并表现出正介电泳（DEP），将细胞移动到电极边缘。然后由于施加在两半电池上的力，细胞被拉伸，导致单轴拉伸，称为电形成。通过改变激励波的振幅，可以很容易地调整剪切应力的水平和由此产生的变形。这样就可以量化红细胞的非线性变形性，以响应高通量下的小变形和大变形。使用ASK策略修改激励波可诱导具有可编程负载速率和频率的循环电形成。这为表征红细胞疲劳提供了一种方便的方法。我们的ASK调制电形成方法首次能够直接测量循环载荷引起的红细胞疲劳。它可以用作一般生物力学测试的工具，用于分析其他细胞类型和患病条件下的细胞变形性和疲劳，也可以与控制细胞微环境的策略相结合，例如氧张力和生物和化学线索。

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.

当细胞悬液加载到微流体通道中时，观察到细胞的相对均匀分布。在函数发生器的信号输出（例如，简单的正弦波或ASK的键控相位）上，薄膜指叉电极产生非均匀的交流电场。悬浮细胞自发地响应这种电激发并表现出积极的DEP行为，即向具有较高场强的电极边缘移动。因此，细胞沿着电极的边缘排列，并且由于电形成而被拉伸。在键控阶段，红细胞由于电形成而拉伸;在非键化阶段，RBC 放宽（<stro...

Watch this Scientific Journal Video about Studying Biomechanics of Circulating Cells by Modulating Their Electrodeformation Behavior at JoVE.com

这里介绍的是使用调幅电形成方法对人类红细胞进行机械疲劳测试的方案。这种通用方法可用于测量悬浮液中生物细胞形态和生物力学特性的系统变化。

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

我们的研究重点是研究循环细胞（如人类红细胞）的细胞生物力学。我们利用电动、微流体和材料科学来了解细胞膜损伤的机械起源，以及某些疾病中血细胞寿命缩短的机制。生物细胞的疲劳研究具有挑战性。它要求对细胞膜施加循环载荷并跟踪单个细胞的变形。使用ASK的振幅表键来调节红细胞的电极变形行为，我们可以量化细胞变形性如何与加载周期保持一致。我们首次证明，红细胞的膜可以单独通过循环拉伸降解。我们的方案利用介电泳将电池移动到电极边缘进行电极变形测量。它不需要任何稳定技术，可以直接用于细胞悬浮。使用叉指微电极使我们能够在单个视场中测量数十个细胞。

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Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Unit 1

Operational Amplifiers

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1.0K 次观看.  Florida Atlantic University. 我们的研究重点是研究循环细胞（如人类红细胞）的细胞生物力学。我们利用电动、微流体和材料科学来了解细胞膜损伤的机械起源，以及某些疾病中血细胞寿命缩短的机制。生物细胞的疲劳研究具有挑战性。它要求对细胞膜施加循环载荷并跟踪单个细胞的变形。使用ASK的振幅表键来调节红细胞的电极变形行为，我们可以量化细胞变形性如何与加载周期保持一致。我们?...

视频: 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 

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.

科研

生物工程

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.

介电泳介质和电变形素材的制备

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.

红细胞电变形微流控装置的制备

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.

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

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.