作者要感谢劳埃德翁建设性投入此技术的早期版本中，杰里米Agresti压力盖设计技巧博士和齐东平博士，他在制造压力盖的帮助。我们感谢M. Teitell和P. Gunaratne的实验室进行测试，提供各种细胞样本。我们感谢美国国家科学基金会（职业奖DBI-1254185），加州大学洛杉矶分校琼森综合癌症中心和加州大学洛杉矶分校临床和转化科学研究所支持这项工作。

1，微流控装置的设计注:该设备设计有四个基本功能区域:输入端口，细胞过滤器，节流阵列，和出口（ 图1）。整体的设计可应用于细胞类型的广泛，与微调尺寸。这里提供的是连同有效的选择主要和永生化细胞的设备参数的一些基本设计建议。 <ol><li>选择缩颈阵列通道（ 图1B）的宽度为平均泡孔直径的约30-50％;此缩颈至细胞大小比结果显著细胞变形，但最小的堵塞。由于以前没有经过测试的细胞类型，谨慎的做法是设计一系列收缩宽度从大约30-50％的细胞直径找到最佳宽度。与许多类型的微流体装置中，超过120设备的模具师傅可以在罪恶图案GLE 4英寸硅片，使不同的设备设计的阵列来制作在一个单一的制造工艺。 </li><li>选择的信道的高度是细胞直径的至少50％;这确保了细胞被限制在2个维度。为了防止焊接过程中通道坍塌，确保渠道的纵横比不小于1点10分（高:宽）整个设备。对于渠道必须超过这个长宽比，增加支撑柱，防止坍塌。空间支撑柱的距离至少是两倍的泡孔直径，以不妨碍信元流。 </li><li>包括过滤器在入境口岸，除去杂物和分解细胞簇（ 图1B）。调整的滤波器的帖子的尺寸和间隔，使得分离的职位的距离大约等于一个单元的直径。 注:特征尺寸的下限是由在其中的光刻掩模被印刷分辨率设置。确保所有的功能都在水库内由掩模供应商指定的olution限制。 </li></ol> 2，用品及准备注:在开始任何实验前，下列项目必须有所准备。整个安装的示意图给出了如图1所示 。 <ol><li>使用标准技术的光刻微14,15制造设备的主人。验证用轮廓的图案特征的高度。 注意:当主人可以很容易地制作在洁净室的设施，一些实验室已经开发出廉价的光刻方法用在半清洁的环 ​​境16,17。 </li><li>准备气源压力驱动的流动，用压缩空气和空气调节器及配件（ 图1A）序列的坦克。 <ol><li>设置的空气供给罐或手动调节。 注:在5％CO 2的组合物在空气中保持类似的环境作为典型的细胞培养incubatoR，但其它气体的混合物也可以使用。 </li><li>设立网上电子控制压力调节器与手动调节。使用一个电 - 气动转换器，调节和维持整个信道的稳定的压力降，以小于2千帕的压力波动。使用LabVIEW编写简单的代码（补充资料）输入所需的压力。 注:电 - 气转换器使用一个内部反馈环路调节阀出口压力在整个微流体装置在指定的压力相匹配。 </li><li>设置一个压力室来驱动流动的细胞悬浮液。组装的细胞悬浮液室走出一个标准的流式细胞仪管，创建于管的压力，密封机械加工的上限。 注:在压力室盖包含两个孔:一个连接到压缩空气罐的入口和一个出口，通过它从加压室的细胞流入装置（ 造型玩具E 2）。 </li></ol></li><li>设置在倒置显微镜配备有相机具有每秒至少100个帧的采集速率来捕获流经缩颈阵列的单元的图像。接口摄像头的视频采集卡和计算机。 </li><li>制备10％的股票的表面活性剂溶液重量/重量的Pluronic F-127的磷酸盐缓冲盐水（PBS）中，添加少量的F127与细胞培养基溶液将有助于减少细胞粘附到PDMS墙壁。轻轻地在37℃下制备的F127溶液，涡流和加热以溶解F127。在使用前，通过0.2微米的针头过滤器过滤该溶液并储存在室温下。事先准备的涡旋和滤波的表面活性剂溶液会产生气泡，这将持续超过24小时。 </li></ol> 3，微流体设备制造<ol><li>制造与微流体通道的PDMS块。 <ol><li>混合硅橡胶已仔细1:10 Ta比（W / W）的固化剂，以碱。 </li><li>倒在设备主（步骤2.1）的混合物。德加在钟罩与应用真空，直到残留气泡消失，或约10-20分钟。 注意:在此时间后，可能仍存在的气泡在气 - PDMS接口，这是正常的;这些在烘焙过程中通常会消散。这是最关键的，以确保有相邻的信道没有剩余的气泡。 </li><li>烘烤脱气装置在65℃下搅拌4小时。为了防止信道塌陷，烘烤设备过夜，以进一步交联的PDMS的设备有较高的弹性模量大于信道塌陷更有抵抗力。但是，请注意延长烘烤也脆化的PDMS，并可能导致冲孔（步骤3.3）时，开裂。 </li></ol></li><li>从母模中取出的微流体装置。切成单个的微流体装置进行的PDMS块用刀片和从桅杆移除装置呃。开始轻轻提起关闭该设备的一个角;缓慢而温和的剥落，而不是解除PDMS块直线上升，降低了双方的PDMS和主应力。 </li><li>打孔的PDMS设备的油管和微通道之间的连接建立连接的端口。建立使用活检穿孔，并从通道侧，通过对所述装置的外侧冲头。 注:活检穿孔带0.75毫米缸径与聚醚醚酮产生漏洞，接口完全醚酮（PEEK），管（外径= 32"为0.79毫米）或聚乙烯管（PE-20，外径= 0.043"或1.09毫米）。使用环形灯，以帮助可视化装置孔，浅通道18。请务必活检冲头锋利;在重复使用后的切削刃变钝和活检穿孔应丢弃并更换。 </li><li>冲洗的PDMS设备用异丙醇（色谱纯），除去硅橡胶的防尘，提出块。确保打孔是明确的碎片从挤压瓶通过孔指导的纯异丙醇源源不断。吹干以过滤空气。清洁后，将硅橡胶块的渠道面对在一个干净的培养皿中，以保持设备清洁。如果需要的话，存储设备在这个形态直到粘接。 </li><li>清洁用甲醇（HPLC级）漂洗该玻璃基板上，吹干燥用过滤后的空气，并将其放置在一个200℃的热板中为5-10分钟，以确保玻璃是完全干净的，干燥的等离子处理之前。 注:将玻璃基板形成各个信道的底部。通常载玻片足够，因为10X或20X的目标是理想的成像多渠道并行。对于更高分辨率的成像，结合该设备盖玻片（＃1.5厚度）。 </li><li>的PDMS装置粘接到玻璃基板上。 <ol><li>氧等离子体处理的PDMS块的信道侧和载玻片的1边。 注:W第i个手持放电单元中，使用1分钟的处理时间，但优化处理时间为每个氧等离子体装置。 </li><li>处理后立即将PDMS的玻璃板上的处理过的一侧的顶部的通道侧。 </li><li>轻压力〜抱团10秒，以促进结合。烘烤在升高的温度下（60-80℃）下至少15分钟，整个装置，以提高粘接强度。 </li></ol></li></ol> 4，通过狭窄的通道形变细胞<ol><li>检查在显微镜下微流体装置。确保该信道没有被折叠或破损的和，这两个入口和出口孔直接连接到设备信道通过使用一个低功率物镜（10X）。通过计分用刀片将PDMS的表面缺陷的指定设备，并且不使用他们的实验。 </li><li>制备的细胞培养物的样品进行测定。使用标准条件培养细胞。 <ol><li> ˚F或例如，培养HL-60细胞在RPMI-1640培养基补充有37℃的1％青霉素-链霉素溶液和10％胎牛血清，5％CO 2的培养箱中培养。 </li><li>对于贴壁细胞，用胰酶消化，重悬在新鲜培养基收获它们。使用标准的胰蛋白酶协议;例如，添加约0.5 ml胰蛋白酶到烧瓶中，用25 平方厘米的文化区，并悬浮在〜30毫升新鲜培养基。以每1×10 5个细胞/ ml 5μM的终浓度如前所述19,20分化的HL-60细胞分化为粒细胞型细胞的全反式维甲酸（ATRA）。 </li><li>离心将细胞以300×g离心3分钟，小心吸去上清，重悬细胞，在新鲜培养基中的适当的最终密度为0.1％体积/体积的F-127。 </li></ol></li><li>计数用库尔特计数器，血球细胞，或流式细胞仪。调整细胞悬浮液以1×10 6〜5×10的密度<s达&gt; 6个细胞/ ml。当悬浮液密度设定，补充以大约0.1％体积/体积的F-127。 注:细胞密度和表面活性剂的浓度，可能需要根据特定的细胞系统中进行修改; 表1提供了以前使用的细胞和F-127的不同类型的细胞浓度的记录。 </li><li>放置细胞悬浮于流式细胞仪管，并连接到压力盖。之前的管道连接到所述微流体装置，将压力调整至约14〜21千帕，并冲洗，直至细胞悬浮液出现从管道的前端。这将有助于最大限度地减少气泡的管道和设备。 </li><li>插入含有细胞悬浮液到装置入口的管线的末端。如果该装置被结合到盖玻片，放置在平坦的固体表面上的装置，例如连接管之前的显微镜载物台;盖玻片是脆弱的，并且可以以其他方式损坏。 </li><li>插入管件的INT澳出口和路线到空管，用于收集废物如空Falcon试管贴在显微镜载物台侧。在实验之间的装置的总流体阻力的一致性，确保入口和出口管道是每个实验相同的直径和长度大致相同的。 </li><li>轻轻地斜坡上升的压力至约28千帕，或者直到细胞流过的通道。定位装置，使多个信道是在视场和垂直于屏幕的底部。如果相机是通过数据大小的限制，获得多个视频生成独立的数据点数量充足。根据需要调整为所需的数据输出的帧速率。为了捕捉改变细胞形态，使用高速成像在每秒300帧（fps）;测量细胞的转运时间，可使用约100帧的帧速率。 </li></ol> 5，数据分析<ol><li>打开文件肥大er_script.m（可下载的补充文件），然后运行该程序。 </li><li>选择第一个视频是从出现在Windows资源管理器窗口进行分析。指定的帧速率选择的视频，然后按Enter。 注:图中就会出现，提示用户选择一个矩形裁剪窗口的第一个视频。 </li><li>选择包括从左至右的所有通道和交叉的通道的正上方的顶部和底部（ 图3）通道的阵列的第一个灯泡的窗口。 </li><li>从裁剪图像选择收缩区域。要特别注意保持选定的每个视频顶部和底部的窗口边界之间的固定像素的距离。 注:上面的窗口边指定最上层收缩和底部的窗口边指定最底部的收缩（ 图4）;中间缢从这个用户的输入近似。接着，该算法显示的Segmentation细胞的每个视频的第一帧50（ 图5）。 </li><li>监视器的左上角图像（ 图5A）紧密，以确定该算法是精确定位的细胞;的二值化图像被叠加在该源视频演示标识单元格的位置。 注:不同的窗口（ 图5B-5D）是代码的诊断和论证后，特定的图像处理操作的视频帧。 </li><li>选择下一个视频可以从Windows资源管理器窗口进行处理。 注:该算法将重复步骤5.2-5.5为每个选定的视频。 </li><li>选择取消，一旦所有想要的影片已通过Windows资源管理器窗口中添加了指示功能的视频列表完成。 注:该算法将没有用户干预的执行剩余的操作。这将需要大约1-2小时，具体取决于视频处理，计算机PE数量rformance。建成后，该算法会产生在途时间数据的直方图，并将数据保存在目录中的一个MATLAB .MAT文件和一个Microsoft Excel xls档案。 </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+Acid+Differentiation+of+HL-60+Cells+Promotes+Cytoskeletal+Polarization.">Retinoic Acid Differentiation of HL-60 Cells Promotes Cytoskeletal Polarization.</a> Experimental Cell Research. 254 (1), 130-142 (2000).</li><li>Rosenbluth, M. J., Lam, W. A., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Microscopy+of+Nonadherent+Cells:+A+Comparison+of+Leukemia+Cell+Deformability+.">Force Microscopy of Nonadherent Cells: A Comparison of Leukemia Cell Deformability .</a> Biophysical Journal. 90 (8), 2994-3003 (2006).</li><li>Tsai, M., Waugh, R., Keng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+HL-60+cell+deformability+during+differentiation+induced+by+DMSO.">Changes in HL-60 cell deformability during differentiation induced by DMSO.</a> Biorheology. 33 (1), 1-15 (1996).</li><li>Rowat, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Envelope+Composition+Determines+the+Ability+of+Neutrophil-type+Cells+to+Passage+through+Micron-scale+Constrictions.">Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions.</a> Journal of Biological Chemistry. 288 (12), 8610-8618 (2013).</li><li>Lam, W. A., Rosenbluth, M. J., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+exposure+increases+leukemia+cell+stiffness.">Chemotherapy exposure increases leukemia cell stiffness.</a> Blood. 109 (8), 3505-3508 (2007).</li><li>Bhattacharya, S., Datta, A., Berg, J. M., Gangopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+surface+wettability+of+poly(dimethyl)+siloxane+(PDMS)+and+glass+under+oxgen-plasma+treatment+and+correlation+with+bond+strength.">Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxgen-plasma treatment and correlation with bond strength.</a> Journal of Microelectromechanical Systems. 14 (3), 590-597 (2005).</li><li>Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+poly(dimethylsiloxane)+surface+modification+to+control+cell+adhesion.">Simple poly(dimethylsiloxane) surface modification to control cell adhesion.</a> Surface and Interface Analysis. 41 (1), 11-16 (2009).</li><li>Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+Microfabricated+Valves+and+Pumps+by+Multilayer+Soft+Lithography.">Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography.</a> Science. 288 (5463), 113-116 (2000).</li></ol>

作者有没有利益冲突披露。

下面我们来分析细胞的使用压力驱动流过收缩的微流体通道过境的变形提供了全面的实验过程。用MATLAB脚本实现自动数据处理（补充材料）;该代码的更新版本保持（ <a href="www.ibp.ucla.edu/research/rowat" target="_blank">www.ibp.ucla.edu/research/rowat</a> ）。更广泛地说，这里提出的技术可以适用于许多基于细胞的微流体分析，包括细胞骨架的24,23的作用和核硬化剂以及测定癌细胞类型4,5的变形。?...

改变细胞形状在许多生物环境是至关重要的。例如，红细胞和白细胞的变形通过毛细血管比自身直径小1。在转移，癌细胞必须变形，通过狭窄的缝隙间，以及曲折的血管和淋巴管网的种子在辅助站点2。探测单个细胞的物理特性，微流控设备提出了可自定义，研究了一系列的细胞行为，包括他们的迁移，通过狭窄的缝隙3，通过微米级的收缩3-9被动变形能力的理想平台。聚二甲基硅氧烷（PDMS）微流体装置是光学透明的，从而使电池变形，以使用光显微镜观察，并使用基本的图像处理工具进行分析。此外，收缩阵列可以被精确定义，同时使多个细胞的分析与吞吐量超过许多现有的技术10,11。 在这里，我们提出了一个详细的实验方案，用于探测使用"细胞变形器"的PDMS微流体装置，细胞变形。该设备被设计成使得通过连续收缩的细胞通路;这种几何结构是在生理环境下，如肺毛细血管床12共同。为了测量细胞变形，运输时间提供了一个容易测量通过一个单一的收缩4,6需要一个单独的单元格过境时的方便度量值。为了保持整个狭窄通道在细胞中转恒定的压力降，我们用压力驱动流。我们的协议包括由压力驱动的流动，制备和细胞的成像，以及图像处理的装置的设计和制造中，设备操作的详细说明，测量时间为细胞变形，通过一系列收缩的。我们有这两个设备的设计和视觉数据处理代码的补充文件。随着数据的代表性样本，我们证明细胞转运时间通过一系列收缩的收缩作为传代次数的函数。时间刻度为细胞转运分析，虽然微流体装置的窄收缩可以揭示在各种细胞类型4,5,13的变形差异。这里展示的设备唯一调查了细胞转运通过一系列的微米级收缩;此设计模拟在循环的细胞经历，也使探测单元的额外的物理特性，如松弛时间的曲折路径。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pluronic F-127 Block Copolymer Surfactant&#160;</td>
			<td style="width:71px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">8409400</td>
			<td style="width:167px;">Produced by BASF, also available through Sigma</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">PDMS base and crosslinker</td>
			<td style="width:71px;">Essex Brownell</td>
			<td style="width:119px;">DC-184-1.1</td>
			<td style="width:167px;">Product commonly named Sylgard 184 Elastomer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Oxygen plasma discharge unit</td>
			<td style="width:71px;">Enercon</td>
			<td style="width:119px;">Dyne-A-Mite 3D Treater</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Biopsy Punch, Harris Uni-Core (0.75 mm)</td>
			<td style="width:71px;">Ted Pella, Inc.</td>
			<td style="width:119px;">15072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight Ferrule, 1/32&#34;</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-113</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight III Fitting, 10-32</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-300X</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyetheretherketone (PEEK) tubing, outer diameter = 1/32&#34;or 0.79 mm</td>
			<td style="width:71px;">Valco</td>
			<td style="width:119px;">TPK.515-25M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyethylene (PE-20) tubing, 0.043&#34; or 1.09 mm</td>
			<td style="width:71px;">Becton Dickinson</td>
			<td style="width:119px;">427406</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pressure regulator</td>
			<td style="width:71px;">Airgas or Praxair</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Polyurethane tubing, 5/32&#8221; OD</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5648K284</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Push-to-connect fittings</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5111K91</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Voltage to Pressure (E/P) Electropneumatic Converter</td>
			<td style="width:71px;">Omega</td>
			<td style="width:119px;">IP413-020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">16-bit,250 kS/S, 80 Analog Inputs Multifunction DAQ</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">NI PCI 6225-779295-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Analog Connector Block-Screw Terminal</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SCB-68-776844-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">LabView System Design Software</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Matlab Software</td>
			<td style="width:71px;">The MathWorks, Inc.</td>
			<td style="width:119px;">Matlab R2012a</td>
			<td style="width:167px;">Code requires the Image Processing Toolbox</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:216px;">Shielded Cable</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SHC68-68</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic technique to probe cell deformability

在这里，我们详细的设计，制造和使用的微流体设备的评估，以有效地对大量单个细胞的变形能力。通常情况下，可以在1小时实验中获得了10〜2单元中的数据。一种自动图像分析程序使图像数据的高效后实验分析，使处理是在几个小时内完成。我们的设备的几何形状是独特的，因为细胞必须变形，通过一系列的微米尺度收缩，从而使初始变形和各个单元的时间依赖性舒张待测定。此方法对人早幼粒细胞白血病（HL-60）细胞的适用性论证。驱动单元发生变形，通过使用压力驱动的流动微米尺度收缩，我们观察到人类早幼粒细胞（HL-60）细胞中，通过随后的收缩传代更快之前暂时阻塞所述第一收缩为9.3毫秒的平均时间离子，每收缩4.0毫秒的平均中转时间。与此相反，全反式视黄酸处理的（嗜中性粒细胞型）的HL-60细胞通过随后的收缩传代以3.3毫秒的平均传输时间之前，闭塞第一缩颈为只有4.3毫秒。这种方法可以更深入地了解细胞的粘弹性性质，最终揭示这种行为的分子起源。

研究了不同类型的细胞，人髓性白血病细胞（HL-60），区分中性粒细胞，小鼠淋巴细胞的细胞，和人卵巢癌细胞系（OVCAR8，HEYA8）的变形用"细胞变形器"微流控技术进行评估。对HL-60和中性粒细胞型HL-60细胞的在途时间代表结果表明，该时间表的单细胞中转通过一系列收缩的， 如图6。运输时间测量单个细胞的人口每7微米的收缩在一系列7收缩在28千帕（ 图6）的驱动压力。 </...

Watch this Scientific Journal Video about A Microfluidic Technique to Probe Cell Deformability at JoVE.com

A Microfluidic Technique to Probe Cell Deformability

我们展示了一个微流体为基础的检测通过微米级的收缩序列来测量时间刻度为细胞转运。

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

a-microfluidic-technique-to-probe-cell-deformability

Research

Education

JoVE Journal

JoVE Core

Statistics

Bioengineering

Unit 1

Measure of Central Tendency

SCIENTISTS IN ACTION

11.2K 次观看.  University of California, Los Angeles. 我们展示了一个微流体为基础的检测通过微米级的收缩序列来测量时间刻度为细胞转运。 

视频: A Microfluidic Technique to Probe Cell Deformability

Measurement of Bioelectric Current with a Vibrating Probe

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 &mu;m), which can detect ionic currents in the &mu;A/cm2 range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 &mu;A/cm2. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

生物电电流测量振动探头

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

科研

生物工程

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.