Name: electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip
Uploaded: 2015-12-29T15:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1.设计和MDF芯片的制备<ol><li>绘制使用商业软件，如AutoCAD单个丙烯酸系层的图案，并保存该图形。 <ol><li>回顾在图1A中的四层压克力板图案的设计和确认层间的连接。 </li></ol></li><li>制造所有的丙烯酸类片材，并通过激光烧蚀使用CO 2激光划线器（图1B，2A 和 2B）的双面胶带。16 <ol><li>打开电池激光划线和机器连接到控制笔记本。 </li><li>运行的商业软件，通过点击打开设计步骤1.1模式"设计图案的文件。" </li><li>将一块空白丙烯酸系片材或双面胶带上的激光划线器的XYZ台。 </li><li>设置激光束的焦点的丙烯酸系片材或双面胶带具有自动alignmen的表面上通过激光划片的制造商提供吨棒。 </li><li>发送所设计的图案，以该激光划线器的丙烯酸系片材或双面胶带的直接加工。 </li></ol></li><li>使用镊子从丙烯酸系片材除去保护纸和吹表面的清洁氮气。 </li><li>堆叠的丙烯酸系片材，并在一个热粘合机在2kg / cm 2的压力结合在一起在110 45分钟 °C至形成流/电刺激通道组件。 </li><li>准备在显微镜盖玻璃作为在芯片中的细胞培养基材。 <ol><li>把防护玻璃成染色缸和填充罐用稀释10倍的物质列表中列出的洗涤剂。 </li><li>把盖玻璃和染色缸中的超声波免缝滤清器并清洁罩玻璃15分钟。 </li><li>倒入稀释的洗涤剂出来的染色缸中，加注蒸馏水罐子，并重复步骤1.5。2 3次。 </li><li>通过将其附着在双面胶带前用氮气吹干燥清洗玻璃盖。 </li></ol></li><li>附着的清洗玻璃盖与双面​​胶带构图步骤1.2的流量/电刺激通道组件。 </li><li>坚持在中纤板芯片组装与超级胶水的第1层13件亚克力适配器连接到单独的开口。密度板芯片组装就完成了（ 图2D）。 </li><li>与使用前紫外线照射30分钟消毒完整的中纤板芯片组装。 </li></ol> 2.设置密度板芯片的盐桥网络<ol><li>在使用之前，消毒所有的塑料管，用手指拧紧螺母， 并且在图3B中所示的离心管通过设置的15分钟的保持时间在121 °下在高压釜中。 </li><li>经由介质入口连接氟塑管（ 图3B-ⅰ）到MDF芯片组件并且，如图1A所示插座适配器。 </li><li>连接介质入口的Luer锥形和出口塑料管在图3B-i到所述旋塞3路。 </li><li>取2.5 ml的CO 2的-equilibrated磷酸盐缓冲盐水（PBS）的使用3毫升注射器和注射器连接到入口塑料管的活塞3路。空3毫升注射器连接到插座的塑料管的活塞3路。这两个注射器通过MDF芯片从而互连。 注:孵育PBS在37 °C 5％-CO 2细胞培养孵化器O / N来获得二氧化碳 -equilibrated PBS。 </li><li>封蓝色的开口和绿色适配器（图1A）上的第1层PMMA板用白色固体手指拧紧螺母（图3B-ⅲ）。 </li><li>与CO 2填充盐桥通道和培养室中设置步骤2.4中所述的注射器-equilibrated PBS。避免形成气泡。 </li><li>接下来，把CO 2 -equilibrated PBS含在37℃，5％-CO 2细胞培养孵化器的O / N培养MDF芯片。这允许在双面胶带的溶解空气，以形成在所述腔室的泡沫。 </li><li>冲洗掉在通道中的气泡，通过使用两个注射器快速PBS流动的通道。泵的PBS来回如果必要的。 </li><li>漏的PBS中通过3路活塞连接到所述介质出口管的通道。 </li><li>使用一个新的3-ml注射器，取2.5毫升的 CO 2 -equilibrated Dulbecco氏改良的Eagle培养基（DMEM）（见步骤3.5），并更换连接到用新的入口的注射器。笔芯渠道与网上平台。 </li><li>制备盐桥网络。 <ol><li>时间上去除固体螺母（ 图3B-III）绿色适配器（ 图1A），注入3％热（&gt; 70℃）的琼脂糖放入盐BRI通过开口中的绿色的适配器（图1A）DGE通道。 注意:热琼脂糖制备:将1.5克琼脂糖粉末在50毫升PBS中。通过在高压釜设定20分钟的保持时间在121℃灭菌。 </li><li>停止注射琼脂糖当液体充满四分之三的盐桥通道的长度的。 </li><li>通过琼脂糖注射后拧固螺母（图3B-ⅲ）密封在绿色适配器（图1A）的孔。 </li><li>替换在蓝色适配器（图1A）与半透明管状手指拧紧螺母（图3B）的固体坚果。 </li><li>加载3％预热琼脂糖入管状螺母（图3B-ⅳ）。 </li><li>嵌入前的银/氯化银电极（图3B-v）的入管状螺母（图3B-ⅳ）凝固的琼脂糖。 </li></ol></li><li>完成盐桥网络的设置完成后，注入CL1-5肺癌细胞到培养腔室，一个腔室的时间。 </li></ol> 3.准备癌细胞，并设立Electrotactic实验<ol><li>肺癌细胞株CL1-5经常文化。 <ol><li>培养的CL1-5肺癌细胞，从教授杨泮池，17中的完全培养基中，在37中得到一个75T细胞培养烧瓶 °下在5％CO 2的气氛中。完全培养基由DMEM和10％牛胎儿血清（FBS）的。子培养细胞每3至4天。用于执行electrotaxis实验的细胞是从原始来源小于25代。 </li></ol></li><li>加入2毫升0.25％胰蛋白酶缓冲器向指数生长的细胞CL1-5并孵育在37 2分钟 °C 5％-CO 2细胞培养孵化器电池detachmen吨。 </li><li>终止该拆卸过程用6ml 10％FBS的DMEM，并离心将细胞在300克5分钟，室温。然后丢弃培养基和悬浮细胞沉淀用5毫升PBS中。 </li><li>计数细胞，在PBS中的数目。然后取1×10 6个细胞，并离心以300g为5分钟，在室温。 </li><li>丢弃的PBS。悬浮细胞用预热的 CO 2 -equilibrated的DMEM，并调整细胞密度为1×10 6个细胞/ ml。 注:在孵育37完全培养基 °C 5％-CO 2细胞培养孵化器O / N来获得二氧化碳 -equilibrated DMEM。 </li></ol> 4.设立Electrotactic实验<ol><li>从密度板芯片的出口处，注入0.3毫升的细胞，1×10 6 /毫升，进入芯片。 </li><li>孵育在细胞培养孵化器的细胞接种密度纤维板芯片（设置在37 ℃和5％的CO 2）为2-4小时。 </li><li> MDF微设置流体系统。 <ol><li>安装MDF微流体系统在透明的氧化铟锡玻璃（ITO），加热器。测量密度板芯片用K型热电偶密度板芯片与ITO玻璃之间夹住的温度。控制在ITO加热器与一个比例 - 积分 - 微分（PID）控制器。设置MDF微流体系统孵化温度为37 °下与PID控制器。 注:在ITO加热器的制造和细胞培养加热系统的安装是在以往的报告18,19说明。 </li><li>安装在倒置显微镜上的计算机控制的XYZ电动载物台的温度控制的中密度纤维板芯片组件。电动载物台的使用方法在步骤5.1中描述。通过使用四通道注射泵入口泵完全培养基向培养腔室。 </li></ol></li><li>泵完全培养基到MDF芯片以20微升/小时的流速。从出的文化浪费让被收集在离心管（示出为图 3A中的"废物"）。 </li><li>在MDF中芯片孵育将细胞再16-18小时，在37℃。 </li><li>要更换介质，泵含有50，25，第5和0μM激酶抑制剂，分别培养基，向每个培养室在20μL/分钟，持续10分钟的流速。 注:加入3毫升无菌水到10.6毫克激酶抑制剂准备Rho相关蛋白激酶抑制剂Y27632的10mM储备溶液。稀释10mM储备溶液至50，25和5μM，分别使用10％FBS的DMEM培养基中。 </li><li>孵育细胞中的激酶抑制剂另外60分钟，在37℃。设置介质流速至20微升/小时。使用相同的培养温度和流量在后面的步骤上述率。 </li><li>使用电线到直流电源连接到经由芯片上的银/氯化银电极密度板芯片。 </li><li>串接电流表我n中的电路。使用电流表监测中的电流密度纤维板芯片。 </li><li>接通直流电源和通过调节供电电压以15至19五设置电流表86.94μA的电流 注意:考虑欧姆定律E = I /（σAEFF），其中I是电流流过块状材料， 即 ，在electrotactic室中的培养基，σ（= 1.38Ω-1米- 1）是培养基中，甲EFF（= 0.21毫米2为3毫米宽×0.07毫米高）的导电率是electrotactic腔的有效截面面积，并且电流（I）中，以产生一个300毫伏/毫米所需EF中密度板芯片是86.94μA。 </li></ol> 5.采集和细胞图像分析<ol><li>控制使用MATLAB GUI程序的机动阶段。通过安装在microsco密度板微流体系统PE，移动芯片采用电动舞台的观察区域。 </li><li>安装在显微镜用数字单镜头反光（SLR）相机记录细胞图像。 </li><li>以使用4X物镜以15分钟的间隔2小时的显微图像。 </li><li>细胞迁移分析。 <ol><li>运行NIH ImageJ的1.47 V. </li><li>从初始的距离进行分析，以细胞的质心的最终位置。 </li><li> 进入 "文件"→"导入"→"图像序列"，并导入九张图像细胞迁移分析。第一图像是零时间的结果，最后图象是120分钟的结果。 </li><li>进入" 分析"→"设置测量"，并勾选旁边的" 重心" </li><li>点击" 写意选择"图标。 </li><li>描绘了选择的细胞的边​​缘中的第一图像。 </li><li>进入"编辑"→ </em&gt;"选择"→" 添加到管理器" </li><li>转到第九图像和描绘在第一图像上所选择的同一细胞的边缘。细胞位置可以使用第二到第八图像进行追踪。 </li><li>进入"编辑"→" 选项"→" 添加到管理器" </li><li>重复步骤5.4.5至5.4.9，以90至100个细胞采集数据。 </li><li>进入" 分析"→"测量"，以获得所选单元格的最初和最后的位置的结果。 注:迁移速度被定义为每小时的平均细胞运动的长度。的指向性被定义为余弦，其中是dcEF（从阳极到阴极）的矢量和从一个单元的起点到其最终位置的向量之间的角度。的指向性是-1细胞向阳极迁移和1对细胞向阴极迁移。对于一组随机心肌梗死光栅单元，所述指向性为0 2 </li></ol></li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+directly+induces+pre-angiogenic+responses+in+vascular+endothelial+cells+by+signaling+through+VEGF+receptors.">Electrical stimulation directly induces pre-angiogenic responses in vascular endothelial cells by signaling through VEGF receptors.</a> J Cell Sci. 117, 397-405 (2004).</li><li>Yao, L., Shanley, L., McCaig, C., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J Cell Physiol. 216, 527-535 (2008).</li></ol>

The authors declare that they have no competing financial interests.

我们发现秉承丙烯酸适配器到MDF芯片的1层是棘手的过程。仅1至2微升的强力胶的应用是足够坚定奉行适配器到MDF芯片。更大量的胶水导致强力胶和不遵守的一个不完整的聚合。一旦丙烯酸适配器牢固地附着在芯片中密度纤维板，液体泄漏的微流体系统很少发生。此外，O / N培养在真空室内有助于去除双面胶带/盖玻璃或双面胶带/丙烯酸片接口之间截留空气。因此，该过程增强了培养室中密度板芯片...

贴壁细胞下的直流电场（dcEF）朝向阳极或阴极移动的行为被称为electrotaxis。细胞的行为electrotactic起着胚胎发生，神经再生，和伤口愈合一个显著作用。1肿瘤细胞如鼠前列腺癌细胞，2-乳腺癌细胞，3和肺腺癌细胞4-8已根据所施加的dcEF所示electrotactic运动。生理EF已在腺组织进行了测量。9,10- Electrotaxis也有报道在腺相关的肿瘤细胞。2,3两者合计，癌细胞的electrotaxis被认为是一个转移因子11控制的电指导dcEF下的癌细胞可以是用于癌症的未来治疗的潜在的方法。然而，今天，electrotaxis的具体分子机制仍存在争议。因此，INF进行调查对癌症细胞迁移的电刺激的luence可以方便的策略的发展为癌症的治疗。 最近，生物微流体装置已被制造用于研究的细胞响应流动的剪切力，12化学梯度，13和电刺激4 体外 。使用聚二甲基硅氧烷（PDMS）或聚甲基丙烯酸甲酯（PMMA，也被称为丙烯酸类）的生物微流体装置的制造已成功地减少了此类实验的故障率。此外，使用基于丙烯酸的微流体装置作为原型调查生物受试者比使用PDMS芯片简单。在丙烯酸​​类装置的各种功能已经开发了用于electrotaxis研究。然而，没有任何先前的设计都能够同时测试的各种化学条件对细胞的影响，并在电场为electrotaxis研究。因此，我们开发了一种微流体装置，所述亩ltichannel双电场（MDF）四个独立培养通道和八个不同的实验条件在一个芯片包含芯片。 丙烯酸基密度板芯片，首先报道了侯等人，8集成电刺激和几个化学隔离通道。这些化学隔离通道可以用来培养在一个实验中不同类型的细胞。所述dcEF在通道是由一电源产生的。两个独立的电场，一用施加的电场强度（EFS）和另一用0 EFS，在各化学分离的信道进行的。以这种方式，在芯片提供了更好的控制的共存EF和化学刺激。此外，从MDF芯片内部的化学扩散数值模拟结果表明，无交叉污染，24小时的试验期后，通道之间发生。8 相比于叠维策报道李等人，14密度板芯片提供了一个更大的文化区域，其允许电刺激的细胞的进一步的生化分析。此外，与中密度纤维板芯片的较大的观察区域，更多的细胞可以在测试中观察，所以迁移速度的电刺激的细胞的或指向性的分析更准确。报告由Huang等人4和Tsai等人。15以前的研究的单通道芯片设计允许被测试仅一种类型的细胞或化学的。然而，中密度纤维板芯片可用于研究的各种化学品上electrotaxis的影响，以及电刺激对不同类型的细胞的作用。换句话说，中密度纤维板芯片允许对化学剂量依赖性的高效学习。

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM medium</td><td>Gibco,Invitrogen, USA</td><td>12800-017</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco,Invitrogen, USA</td><td>16000-044</td><td></td></tr><tr><td>Trypsin</td><td>Gibco,Invitrogen, USA</td><td>25200-072</td><td></td></tr><tr><td>PBS</td><td>Basic Life</td><td>BL2651</td><td></td></tr><tr><td>Y-27632 (hydrochloride)</td><td>Cayman Chemical Co</td><td>10005583</td><td></td></tr><tr><td>agarose</td><td>LONZO, USA</td><td>SeaKem LE AGAROSE</td><td></td></tr><tr><td>syringe</td><td>Terumo</td><td>3 ml with Luer taper</td><td></td></tr><tr><td>3-way stopcock</td><td>Nipro</td><td>with Luer taper</td><td></td></tr><tr><td>PMMA (acrylic)</td><td>HiShiRon Industries CO., Ltd, Taiwan</td><td></td><td>thickness 1mm, 2mm</td></tr><tr><td>acrylic adaptor</td><td>KuanMin Technology Co., Ltd, Taichung, Taiwan</td><td>1/4-28 port, 10x10x6 mm</td><td>customized</td></tr><tr><td>nut</td><td>Thermo Fisher Scientific Inc.</td><td>UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td><td></td></tr><tr><td>Microscope cover glass</td><td>Deckgl&amp;auml;ser, Germany</td><td>24x60 mm</td><td></td></tr><tr><td>double-sided tape</td><td>3M</td><td>PET 8018</td><td></td></tr><tr><td>super glue</td><td>3M</td><td>Scotch Liquid Plus Super Glue</td><td></td></tr><tr><td>TFD4 detergent</td><td>Franklab, France</td><td>TFD4</td><td></td></tr><tr><td>ultrasonic steri cleaner</td><td>LEO ULTRASONIC CO., LTD., Taiwan</td><td></td><td></td></tr><tr><td>Thermo bonder</td><td>KuanMin Technology Co., Ltd, Taichung, Taiwan</td><td>customized</td><td></td></tr><tr><td>CO2 laser scriber</td><td>LTT group, Taiwan</td><td>ISL-II</td><td></td></tr><tr><td>proportional-integral-derivative (PID) controller</td><td>JETEC Electronics Co., Japen</td><td>TTM-J40-R-AB,</td><td></td></tr><tr><td>K-type thermocouple</td><td>TECPEL</td><td>TPK-02A</td><td></td></tr><tr><td>4-channel syringe pump</td><td>KdScientific, USA</td><td>250P</td><td></td></tr><tr><td>DC power supply</td><td>GWInstek, Taiwan</td><td></td><td></td></tr><tr><td>X-Y-Z motor stage</td><td>TanLian, E-O Co. Ltd., Taiwan</td><td>customized</td><td></td></tr><tr><td>inverted microscope</td><td>Olympus, Japan</td><td>CKX41</td><td></td></tr><tr><td>digital SLR camera</td><td>Canon, Japan</td><td>60D</td><td></td></tr></tbody></table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

定向细胞迁移的下一个直流电场（dcEF）的行为被称为electrotaxis。生理dcEF在胚胎发育，细胞分化和伤口愈合过程中引导细胞运动的显著的作用已被证明在许多研究。通过将微流体芯片的electrotaxis测定中，调查过程缩短和实验误差最小化。近年来，微流体装置制成的聚合物质（例如，甲基丙烯酸甲酯，聚甲基丙烯酸甲酯，或丙烯酸）或聚二甲基硅氧烷（PDMS）已被广泛用于研究细胞的响应电刺激。但是，与制造一个PDMS装置所需的许多步骤中，丙烯酸类微FL uidic芯片的简单而快速的结构使得它适合于设备的原型和生产。然而，没有报道的设备，这有助于该组分化学DCE的效率研究对细胞˚F影响。在这份报告中，我们描述我们的设计和丙烯酸系多信道双电场（MDF）芯片调查化学和电刺激对肺癌细胞的效果并发的制造。中密度纤维板芯片提供了一个测试的电气/化学刺激八种组合。该芯片不仅大大缩短了实验所需的时间，但在electrotaxis研究也提高了准确性。

制造中密度纤维板设备和组装 丙烯酸基密度板芯片的示意图示于图1A。四压克力板，一个覆盖玻璃，13丙烯酸适配器，和一块双面胶带在已完成的中密度纤维板芯片（图2D）的组件被使用。只有四个独立的文化通道中密度纤维板设备。然而，芯片上的盐桥网络创建八个不同的?...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.7K 次观看.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

视频: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

神经科学

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

使用交流介电泳分离非转移性（MCF-7）和非肿瘤（MCF-10A）乳腺癌细胞的微流体装置

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using ...

工程学

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

微流体设备中的电场诱导神经前体细胞分化

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

用于分离多功能循环肿瘤细胞的临床微流控芯片平台

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...