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>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SCILOGEX</td>
			<td>911015119999</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>EC Low Conductivity meter</td>
			<td>ecoTestr</td>
			<td>358/03</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160; &#160;Snap-Cap MicrocentrifugeTubes</td>
			<td>www.eppendorf.com</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft&#160;</td>
			<td></td>
			<td>Graph plotting</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>SIGLENT</td>
			<td>SDG830</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass/ITO Electrode Substrate</td>
			<td>OSSILA</td>
			<td>S161</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>OLYMPUS</td>
			<td>IX81 - SN9E07015</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Oven</td>
			<td>QUINCY LAB (QL)</td>
			<td>MODEL 30GCE</td>
			<td>Digital Model</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td>Dassault Systemes</td>
			<td></td>
			<td>CAD software</td>
		</tr>
		<tr>
			<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。

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

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 

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。

调幅电形成评估生物细胞的机械疲劳

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。

amplitude-modulated-electrodeformation-to-evaluate-mechanical-fatigue

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Amplitude-Modulated Electrodeformation to Evaluate Mechanical Fatigue of Biological Cells

Unit 1

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1.4K 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 

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
	Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. 
	We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

小鼠颈动脉的力学性能测试:从新生儿到成人

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

同时进行机电刺激增强细胞心肌成功能的潜能

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

生成层粘连蛋白-111的分形微观结构以向细胞发出信号

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...