This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.

1.芯片设计和制造<ol><li>绘制图案被使用商业软件24的PMMA基板和双面胶带消融。 <ol><li>研究化学浓度和剪切应力的影响，在其每五个培养区（ 图1A和1B）的端绘制具有变化宽度的"圣诞树"的图案。 </li><li>研究化学浓度和电场的影响，绘制了"圣诞树"的图案为盐桥两个流体通道（ 图2A和2B）。 </li></ol></li><li>划线上的聚甲基丙烯酸甲酯片材或通过加载相应文件到的 CO 2激光划线双面胶带单个图案。 <ol><li>打开激光划片机，并检查其与电脑连接。打开模式使用商业软件进行消融。 </li><li>将有机玻璃片或双面胶带Ø■首页划片的舞台。如有必要，使用校准杆和可见的He-Ne激光调节的 CO 2激光的焦点。 </li><li>加载模式进入划片上PMMA板材或带消融。 </li><li>拿起图案片或胶带，删除不需要的部分，并清洗用氮气吹面。 注:聚甲基丙烯酸甲酯片的厚度为1毫米，并且该双面胶带为0.07毫米或0.22毫米。 </li></ol></li></ol> 2.芯片组装<ol><li>与超级胶水，通过对准螺纹和孔在顶部附丙烯酸适配器（长度×宽度×高度= 10毫米×10毫米×5毫米，在中间螺纹）连接到最顶层的芯片 - 大多数层。这些适配器作为介质入口/出口和盐桥连接器。 </li><li>组装层流罩内的微流体芯片。 </li><li>组装化学剪切应力的微流体芯片（CSS芯片）。 <ol><li>一个ttach使用2个刻划双面胶带的划线的PMMA片3张。 （ 图1A和1B）。 </li><li>多一个片0.07毫米厚的双面胶添加到芯片的底部和芯片连接到一个直径10厘米的培养皿。 </li><li>把组件芯片在真空室中过夜。 </li></ol></li><li>为了组装化学电场芯片（CEF芯片），聚甲基丙烯酸甲酯片附着到厚的双面胶带= 0.22毫米（ 图2A和2B）。 <ol><li>附加划线PMMA板和双面胶带，使用此相同的片带的直径10厘米的培养皿。 </li></ol></li><li>离开罩内组装芯片和其暴露在紫外线下消毒30分钟。 </li><li>通过塑料管，并用手指拧紧螺母连接入口，以两个3毫升的注射器。通过塑料管和一个手指拧紧螺母连接插座到废液管。 注:蒸压对于使用前15分钟所有的管子和螺母在121℃。 </li><li>慢慢地压低在注射器素用PBS流体通道的柱塞。推注射器来回去除气泡。 </li><li>把芯片培养箱内过夜，在37℃下，5％CO 2。 注意:这两个步骤的目的是在芯片内洗芯片和除去任何剩余的气泡。 </li><li>就拿芯片出来的孵化器。 </li><li>准备琼脂盐桥在CEF芯片产生的电场。 <ol><li>溶解使用微波在1×磷酸盐缓冲盐水（PBS）缓冲液3％低熔点琼脂糖。 </li><li>注射液分阶段琼脂糖放入盐桥通道和插入电极液凝固之前。 </li><li>放置银/氯化银电极到管状螺母。 </li></ol></li><li>注射器连接到注射泵，不断流动的培养基（Dulbecco改进的EagleR17;培养基，DMEM）中成20微升的流速为10分钟的流体通道/分钟更换PBS中。 </li></ol> 3.细胞制备及实验装置注意:预暖1×PBS中，培养培养基（DMEM加FBS）和胰蛋白酶在使用前37℃水浴中。 <ol><li>板2×10 5个肺癌CL1-5细胞25,26中用DMEM加10％胎牛血清（FBS）的供给的10cm培养皿。孵育培养箱内的细胞在5％ 的 CO 2，在37℃直至90％汇合。 </li><li>吸出培养基并用预热1×PBS中洗一次细胞。 2毫升0.05％胰蛋白酶缓冲液添加至细胞中并等待2至3分钟，在37℃下分离的细胞。 </li><li>转移细胞到15毫升的无菌离心管中，加6 ml培养介质的进入管。轻轻颠倒试管混合并取出5微升含细胞培养基用于在hemocytomete计数细胞数河</li><li>离心300×管 克5分钟。暂停10 6个细胞于1ml培养基中，然后将含有细胞的培养基中，在1毫升注射器。 </li><li>注入含有细胞的介质进入从出口的微流控芯片，并确保该溶液通过五个培养区域分配的所有。孵育培养箱内的芯片下，5％CO 2在37℃下2小时。 </li></ol> 4.实验装置<ol><li>取芯片出保育箱，将其放在一个透明的氧化铟锡（ITO）的玻璃加热器的顶部。 注:ITO玻璃被连接到比例 - 积分 - 微分（PID）控制器，用于通过反馈从热耦合器37±0.5℃，将温度保持在ITO加热器和芯片之间紧紧夹住。 </li><li>把芯片加热器组件上倒置显微镜的电动XY载物台的顶部，用于跟踪细胞迁移或固定XY台倒置荧光显微镜测定ROS的产生。 </li><li>产生不同的化学物质浓度，填充两个注射器用5ml相对浓度0和1（溶解于培养基中）的化学溶液和它们泵入芯片在所需流率:在CSS芯片，以0.3ml的流速/分钟1小时;在CEF芯片，以20微升/分钟的流速在第20分钟，20微升/小时的另一个120分钟（总时间= 2小时）的流速。 </li><li>在CEF芯片，用于跟踪细胞迁移，程序显微镜的XY阶段重复拍照，通过数字单镜头反光文化领域，每15分钟2内（视场）的意见某个字段（DSLR）相机，小时。 </li><li>为了测量产生活性氧的，使用基于荧光的指示器2&#39;- -7&#39;- dichlorodihydrofluoresce二乙酸酯（2&#39;- 7&#39;- DCFDA）。 <ol><li>准备2&#39;-7&#39;- DCFDA的股票在10毫米分子生物学级迪甲基亚砜（DMSO）中。稀DCFDA在DMEM只无血清（5微米的DMEM）。潜在的脱乙酰酶可以增加背景信号，并在细胞减少的信号。 </li><li>在CSS芯片或之后的CEF的芯片的EF刺激的2小时的剪切应力刺激1小时后，泵2&#39;- -7&#39;- DCFDA（5μM在DMEM）到芯片在20μL/分钟的流速为前20分钟和20微升/小时的另外20分钟的流速。用于洗涤，泵的DMEM到芯片在20微升/小时的流速进行20分钟。 </li><li>拍照，通过电荷耦合器件（CCD）照相机，培养区域内的某些视场的，用于分析荧光强度。 </li></ol></li></ol> 5.化学品浓度，剪切应力和电场的计算<ol><li>在这两个CSS芯片和芯片CEF，计算在五个养殖区域的化学浓度。例如，通过注入精矿的H 2 O 2的ations 0并从两个入口200μM，0，25，则产生100，175和200μM的浓度。 注意:通过假定所有的液体分流顺畅，同样左右叉，相对浓度中的五个培养区域分别是0，1/8，1/2，7/8和1。 </li><li>在CSS芯片，采用在每个培养区域的计算的剪切应力（τ） <img alt="式（1）" src="/files/ftp_upload/54397/54397eq1.jpg" /> 27，其中Q是体积流量，η是流体粘度，h是信道的高度，w是所述信道的宽度。 注:通过设定Q = 0.3ml /分钟在每个入口（Q f = 0.12毫升/分钟在各培养区） 中，η= 0.0008帕·秒为培养基中，h = 1毫米，以及w = 1〜4毫米，在剪切应力的计算从0.0048帕（4毫米宽的区域），以0.0192帕（1毫米宽的区域）范围内。 </li><li>在在CEF芯片，计算EF强度中的每个使用E = I /（σAEFF）（欧姆定律），其中I是穿过流体通道中流动的电流的文化区域，σ是培养基的电导率，和A eff是信道的有效横截面面积。 注意:使用σ= 1.38Ω-1 M -1为培养基和A EFF = 0.22毫米2（宽= 1毫米，高度= 0.22毫米）时，EF强度计算为E（毫伏/毫米）= I（ A）×3.3×10 6。 <ol><li> 如图2D所示，治疗的等效电路五C形截面电路用四个（8 + 35 + 8）段和一个（5 + 35 + 5）段。 注:通过分析，根据基尔霍夫电压定律和欧姆定律这一并联电路，在五个文化流动的电流如，I 1到I 5从底部到顶部，被计算为围绕0.49 I（区域1），0.25 I（区域2），0.13 I（区域3），0.08 I（区域4），和0.05 我 （区5），分别，其中I是总的直流（dc）。与0.157毫安，254的EF，130，67，41，和26毫伏/毫米的施加直流被五个培养领域内产生。 注:对于微流体网络的简化电分析中，所有的流体段被认为是与电阻正比于其长度电阻。 </li></ol></li></ol> 6.数据分析注意:使用ImageJ的软件进行数据分析。 <ol><li>分析ROS的生产。 <ol><li>运行该软件的ImageJ。进入"文件"→打开加载一个荧光图像进行分析。 </li><li>转到"图像"→"类型"→"16位"改变了IM年龄为灰度。 </li><li>绘制多边形附上一个单元格。去"分析"→"测量"来测量细胞的平均荧光强度。 </li><li>重复6.1.3从三个独立实验的至少50个细胞收集强度，并计算与平均值的标准误差（SEM）的平均强度。 </li><li>重复6.1.1-6.1.4每个实验条件。 </li></ol></li><li>分析细胞迁移。 <ol><li>运行该软件的ImageJ。进入"文件"→打开加载时间= 0时拍摄的图像进行分析。 </li><li>绘制多边形附上一个单元格。去"分析"→"测量"，测量细胞的质量中心为（x 1，Y 1）。 </li><li>重复6.2.1- 6.2.2测量相同的小区的质量的其它图像的中心为（X 2，Y 2）取在时间= 吨 。 </li><li>计算的迁移速率（以微米/小时）该小区作为<img alt="公式（2）" src="/files/ftp_upload/54397/54397eq2.jpg" /> 。 </li><li>重复6.2.1-6.2.4收集来自三个独立实验的至少50个细胞的迁移率，并计算与平均值的标准误差（SEM）的平均迁移率。 </li><li>从6.2.2和6.2.3，计算该细胞的迁移指向性余弦或θ <img alt="公式3" src="/files/ftp_upload/54397/54397eq3.jpg" /> ，其中θ是施加的EF的矢量之间，并从开始到小区（ 图2G）的端部位置的矢量的角度（从正到负）。 </li><li>重复6.2.6从三个独立实验的至少50个细胞收集迁移指向性，并计算与平均值的标准误差（SEM）的平均迁移指向性。 </li><li>计算用SEM的平均迁移率和用SEM的平均迁移指向性为每个实验条件。 注:指向性+1表示所有细胞向阴极迁移，和-1值表示所有单元向阳极迁移。一组随机移动的细胞的指向性是接近0。 </li></ol></li><li>分析单元格对齐。 <ol><li>运行该软件的ImageJ。进入"文件"→"打开"加载图像进行分析。 </li><li>治疗细胞为椭圆和画一条线，以指示一个单元的长轴线。转到"分析"→"办法"来衡量β线和水平EF方向之间的角度。 </li><li>重复6.3.2从三个独立实验的至少50个细胞收集β，计算平均余弦β与（SEM）平均值的标准误差。 </li><li>重复6.3.1-6.3.3每个实验条件。 注:余弦为+1β表示所有的细胞在平行于所施加的EF对齐，和一个0值指示所有单元对齐perpendicularly以施加EF。 </li></ol></li></ol>

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The authors have nothing to disclose.

PMMA基芯片使用激光烧蚀和写入与基于PDMS芯片，从而需要更复杂的软光刻当它们是便宜和更容易的方法制造。设计的微流体芯片之后，在制造和装配可以只在5分钟内完成。有迹象表明，应注意在执行实验中支付给一些关键的步骤。首先是"组装"的问题。适配器应适当粘在最上面的层的芯片。如果施加太大胶可能泄漏进入流体通道，并且如果使用太少的胶液可能泄漏出来的芯片。另外，在组装的PMMA基板和双面胶带，其将压力施加到压紧整个芯片，以防止任何液体泄漏是很重要的。第二个是"泡沫"的问题。以除去气泡，1×PBS中连续流入1或2分钟的流体通道以20微升/分钟的流速，然后在芯片为p在37℃下被系带培养箱内过夜，5％ 的 CO 2。如果在流体通道内剩余的气泡，该流程将是不均匀的，导致不均匀的化学浓度和EF分布。第三个是"小区负载"的问题。装入培养区域的细胞的数量应被很好地控制。如果细胞数量太少，不少实验运行应当进行收集足够的数据作统计用途。如果细胞数太高，这将是很难区分各个单元和量化其迁移。 在CSS芯片，"圣诞树"结构具有三角形培养面积的总和在五个方面产生不同的化学浓度，每一个都具有一个剪应力梯度。流体剪切应力被称为影响附件，形态学，迁移和细胞的活性。低至0.25-0.6霸接口能细胞附着的剪切应力，报告讲（0.5 -10帕），甚至更高的价值以去除贴壁细胞29。不等的流30，以及甚至更低的值（0.1-1帕）的方向上从0.8到1.5帕诱导的细胞对准层的剪切应力被已知影响细胞形态和渗透率29。此外，受到剪切2-120 Pa的压力平滑肌细胞经历了细胞数量31下降。 Lu 等人 。报告的细胞粘附27定量分析的微流体剪切设备的设计和施工。流体通道内的剪切应力通过改变通道的尺寸进行修改。 Chin 等 。记载的血流动力学上实验室一个芯片上系统，用于控制培养基的流速在微通道以模拟血液的流动剖面在容器32上 。流体通道内的剪切应力通过改变介质的流速改变。在里面本身两个装置，即使可能产生的任何值的剪切应力，只有一个单一的值是一种文化区域之内提供一次。通过比较，本CSS芯片具有提供在一个三角形区域的剪切应力梯度，尤其是增加的实验吞吐量筛选目的的优点。 在CEF芯片，"圣诞树"结构的文化区垂直连接以形成流体路径类似的电路。在施加直流之一，是在培养的区域内产生的，每个具有特定化学浓度的流5 EF强度。按照惯例，electrotactic实验在培养皿中，其中可能发生的EF不精确，中蒸发，大细胞/试剂消耗，并增加了焦耳热的问题进行。封闭的，微型微流体装置有效地克服这些缺点。例如，研究electrotaxisØ˚F人血记忆T细胞，Lin 等人 。报道含有两个相同的，并排的侧微通道，两个改性枪头担任介质水库，以及EF应用15两个铂电极的塑料微流体装置。为了增加吞吐量，PMMA基electrotactic碎片制造为在一个单一的设备12,33提供多种EF强度。此外，能够产生可控的化学和电刺激的微流体装置同时较高的兴趣。 Li 等人 。制作一个基于PDMS微流体设备生成单个或共存化学梯度/ EF文件调查的T细胞14的趋化或electrotactic迁移。 Kao 等。采用类似的微流体装置通过使用EFS 12调节肺癌细胞的趋化性。在这两个装置，一个Y形结构施加在一个单一应用的EF创建稳定的浓度梯度。侯等人 。报告多信道-双电场芯片研究的化学品和EFs对肺癌细胞21并发效应。该装置能够在一个实验中提供电气/化学刺激（2电气×4化学品）的8种组合。虽然可以通过增加，该芯片的能力被限制为（1）只有一个EF强度（加上一个零作为对照），和（2）所需要的泵送化学品为四个独立通道的四注射器泵。通过比较，本CEF芯片具有经由一个单组电极和第2注射泵产生5 EF长处具有五个化学浓度在一起的优点。 尽管这些芯片很容易制造，它们具有一定的局限性。首先，可以产生只有五个"特殊"的浓度和五"具体的"EF优势。第二，该流体通道的宽度不能因的聚焦小于1毫米的 CO 2激光束。但是，通过精确控制在入口与穿过流体通道的电流的化学浓度，任何浓度和EF强度可以达到在培养区域。总之，本CSS和CEF芯片能够提供细胞具有可控单或共存化学/电/剪切应力刺激。在一个单一的CSS芯片，可以产生在与剪应力梯度组合五个不同的化学浓度。此外，在一个单一的CEF芯片，可以创建在有五个EF优势组合五个不同的化学浓度。通过增加"圣诞树"结构的分支，这些芯片的吞吐量可进一步提高。这些芯片被证明是一种简单，省时省力，可靠，高通量平台下的化学/电气/剪切应力刺激细胞行为的研究。

体内细胞通过各种生物分子，包括细胞外基质（ECM），碳水化合物，脂质，细胞和其它细胞的包围。它们官能通过响应微环境刺激如使用ECM相互作用和反应的各种生长因子的化学梯度。传统上， 在体外细胞研究是在细胞培养皿进行，其中细胞和试剂的消耗大，细胞生长在静态（非循环）环境。最近，随着流体部件集成的微制造的器件中的更可控的方式提供了用于细胞的研究的替代的平台。这样的装置能够同时最大限度地减少细胞和试剂的消耗产生的化学和物理刺激的精确微环境。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或polyethylenetere邻苯二甲酸酯（PET）的基板1-3。基于PDMS的设备是透明的，生物相容的，并能透过气体，使它们适合于长期的细胞培养和研究。 PMMA和PET基材是廉价和易于使用激光烧蚀和写作进行处理。 微流体装置应提供细胞与稳定可控的微环境，其中细胞受到不同的化学和物理刺激。例如，微流体芯片被用来研究细胞的趋化性。而不是雇用Boyden小室和毛细管4,5这些微型流体装置能产生精确的化学梯度用于研究细胞的行为1,6,7传统的方法。另一例子是研究细胞的下电场功率（EFs），一个electrotaxis现象名为定向迁移。据报道细胞Electrotactic行为可能与神经再生8，胚胎发育9和伤口愈合10,11。许多研究已经进行了调查各种细胞类型，包括癌细胞12,13的electrotaxis，淋巴细胞14,15，白血病细胞11，和干细胞16。按照惯例，培养皿和盖玻片用于构建electrotactic室产生的EF 17。这种简单的设置姿势介质蒸发和不精确EF的问题，但是它们可以通过封闭的，明确定义的流体通道12,18,19的微流体装置来克服。 为了系统地研究下精确，可控化学和电刺激细胞的反应，这将是很有用处的发展能够同时提供多个刺激细胞的微流体装置。例如，Li 等人 。报告基于​​PDMS-微流体装置，用于创建单个或共存化学梯度和EFS 20。 Kao 等 。开发私奔类似微流体芯片由EF文件6调节肺癌细胞的趋化性。此外，为了提高吞吐量，侯等人 。设计和制作基于PMMA-多声道的双电场芯片，提供细胞与8种不同组合的刺激，是（2 EF优势×4化学物浓度）21。为了进一步增加整个并添加剪切应力刺激，我们开发了两个PMMA基微流体装置根据单个或共存化学/电/剪切应力刺激研究细胞应答。 由Lo 等 22,23报道，这些设备包括五个独立的细胞培养渠道受到持续流体流动，模仿体内循环系统。在第一芯片（化学剪切应力芯片或CSS的芯片），五个相对浓度为0，1/8，1/2，7/8，和1在培养区域生成，并剪切应力梯度是机生产线土木工程署内各五个文化领域。在第二芯片（该化学电场芯片或CEF芯片），通过使用一个单组电极和第2注射泵中，除了这些培养区域内五个不同的化学浓度时，产生五个EF强度。数值计算和模拟执行，以便更好地设计和操作这些芯片，而这些设备内培养肺癌细胞受到单个或共存刺激用于相对于观察到的活性氧物种的它们的响应（ROS），迁移率，和运移方向。这些芯片被证明是节省时间，高吞吐量和可靠的器件用于研究细胞对各种微环境刺激的反应。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-092</td><td>Cell culture medium</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25300-054</td><td>detach cell from the dish</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>10082147</td><td>Cell culture medium</td></tr><tr><td>10 cm cell culture Petri dish</td><td>Nunc</td><td>150350</td><td>Cell culture</td></tr><tr><td>Bright-Line&amp;nbsp;Hemacytometer</td><td>Sigma</td><td>Z359629</td><td>Cell Counting Equipment</td></tr><tr><td>PMMA</td><td>Customized</td><td>Customized</td><td>Microfluidic chip</td></tr><tr><td>Adaptor</td><td>Customized</td><td>Customized</td><td>Microfluidic chip</td></tr><tr><td>0.07/0.22 mm double-sided tape&amp;nbsp;</td><td>3M</td><td>8018/9088</td><td>Microfluidic chip</td></tr><tr><td>Low melting point agarose</td><td>Sigma</td><td>A9414</td><td>Salt bridge</td></tr><tr><td>2&#039;-7&#039;-dichlorodihydrofluoresce diacetate</td><td>Sigma</td><td>D6883</td><td>Intracellular ROS measurement</td></tr><tr><td>Indium tin oxide (ITO) glass</td><td>Merck</td><td>300739</td><td>Heater</td></tr><tr><td>Proportional-integral-derivative controller&amp;nbsp;</td><td>JETEC Electronics Co.</td><td>TTM-J4-R-AB</td><td>Temperature controller</td></tr><tr><td>Thermal coupler</td><td>TECPEL</td><td>TPK-02A</td><td>Temperature controller</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; laser scriber</td><td>Laser Tools &amp;amp; Technics Corp.</td><td>ILS2</td><td>Microfluidic chip fabrication</td></tr><tr><td>Syringe pumps</td><td>New Era</td><td>NE-300</td><td>Pumping medium and chemicals into the chip</td></tr><tr><td>Power supply</td><td>Major Science&amp;nbsp;</td><td>MP-300V</td><td>Supplying direct currents</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>CKX41</td><td>Monitoring cell migration</td></tr><tr><td>Inverted fluorescent microscope</td><td>Nikon</td><td>TS-100</td><td>Monitoring cell migartion and fluorescencent signals</td></tr><tr><td>DSLR camera</td><td>Canon</td><td>60D</td><td>Recording bright-field images&amp;nbsp;</td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-Qi1</td><td>Recording fluorescent images&amp;nbsp;</td></tr><tr><td>super glue</td><td>3M Scotch</td><td>7004</td><td>Attaching adaptors to PMMA substrates</td></tr><tr><td>AutoCAD</td><td>Autodesk Inc.</td><td></td><td>Designing microfluidic chips</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>Dissolving DCFDA</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>Quantifying fluorescent intensities and cell migration</td></tr></tbody></table>

designing microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli

微流体设备能够创建pH值，温度，盐浓度，和其它物理或化学刺激的精确和可控的细胞微环境。他们已经通过像环境在体内提供了通常用于体外细胞研究。尤其，细胞如何响应于化学梯度，电场，应力和剪应力引起了许多利益，因为这些现象对理解细胞特性和功能非常重要。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或聚对苯二甲酸乙酯（PET）衬底。出这些材料，PMMA基底是便宜的，并且可以使用激光烧蚀和书写容易加工。虽然一些微流体装置已被设计和制造用于产生多个共存化学和电刺激，其中没有一个被认为是够在减少实验重复序列，特别是用于筛选目的有效。在这份报告中，我们描述了我们的两个基于PMMA微流控芯片设计和制造用于研究细胞反应，在生产活性氧和迁移，下单或共存化学/电气/剪切应力刺激。第一芯片产生五个相对浓度0，1/8，1/2，7/8，和1在培养的区域，与内部这些领域中产生的剪切应力梯度在一起。第二芯片产生相同的相对浓度，但与各培养区中创建的五个不同的电场强度。这些装置不仅提供细胞与精确，可控的微环境，也大大增加了实验的吞吐量。

化学 - 剪切应力（CSS）芯片 CSS的芯片进行了三次PMMA板，每个的厚度为1毫米，经由两个双面胶带连接在一起，每一个的厚度0.07毫米（ 图1A和1B）的。的"圣诞树"结构产生的0 5相对浓度，1/8，1/2，7/8和1中的五个培养领域。由培养面积设计为三角形，剪切应力梯度，与相关的体积流速，流体粘度和流体通道的尺寸大小，则在每个区域的创建。然后，该芯片被安装，经由另一0.07毫米厚双面胶带，至培养皿培养肺癌CL1-5细胞以及响应于不同的化学浓度和剪切应力，观察到产生活性氧的。在肺癌细胞中ROS的产生是高度相关的路NG癌细胞转移和发展，以及某些化学品和剪切应力被证明参与ROS的产生23。 首先，要研究的H 2 O 2，化学刺激的效果，对ROS的产生，将细胞用2 O 2 的溶液，在0，25，100，175，和200μM连续流动h的温育。如图1C所示，荧光强度的增加作为H 2 O 2浓度的增加，这表明的 H 2 O 2刺激产生活性氧的。接着，为了调查剪切应力对产生活性氧的效果，将细胞暴露于0.0048 Pa的剪切应力梯度0.0192帕。 图1D显示了荧光强度随着剪切应力增加（剪切应力分别为最高分别最低的正面和背面的地区，），建议ING，较高的剪切应力诱导更多的ROS的产生。此外，这个CSS芯片用于研究产生活性氧的响应于不同浓度的α生育酚，维生素E的细胞一种形式的抗氧化剂是由剪切应力加0α生育酚，3.2，12.5，21.9激和25微克/毫升。 如图1E所示，对于α生育酚的浓度低于21.9微克/毫升，荧光强度随着浓度的增加而降低，这表明在降低ROS产生的α生育酚的作用。然而，当浓度升高到25微克/毫升，平均强度也增加了，这表明α生育酚的这种高浓度相比低浓度没有消除大部分的ROS。 化学电场（CEF）芯片在CEF芯片是由1毫米厚的PMMA和0.2的2毫米的双面胶带与图案化在其上（ 图2A和2B）的流体通道。同样，"圣诞树"结构产生的0 5相对浓度，1/8，1/2，7/8和1中的五个培养领域。另外，这五个平行区域被垂直地连接，以形成一个流体路径类似的电路。培养区域内产生的电场有关的流体通道的截面积，和直流（dc）通过的区域的导电性。根据基尔霍夫电压定律和欧姆定律，在五个培养区域中的电流0.49 我 ，0.25 我 ，0.13 我 ，0.08 I和0.05 我 ，分别，其中I是所施加的直流（ 图2C）。该芯片被安装，通过0.22毫米厚的双面胶带，至培养皿培养肺癌CL1-5细胞，观察细胞迁移和生产中的响应于不同的化学浓度和电场的ROS的n个。 首先，该芯片被用来研究ROS的产生针对不同浓度和厚朴酚，EF的不同的优势，两者结合的治疗方法。厚朴酚，小分子多酚从该属厚朴分离，被发现有抗血管生成，抗发炎，抗肿瘤和在临床前研究28的特性。如在图2D中所示，活性氧产生几乎对EFS超过67毫伏/毫米下是相同的，但随着这个值以上的EF增加。 图2E示出了，根据电场和厚朴酚的组合治疗，所述ROS水平住几乎同样的，这表明厚朴抑制外源性产生的活性氧（ 即 ，ROS与EF刺激），尤其是在较高的基本文件。下单或共存化学细胞迁移/电刺激，用这种芯片CEF还研究。如在图2F所示，不加入和厚朴酚，迁移率提高作为EF强度增加。加入不同浓度的厚朴酚后，迁移率一般降低，这表明厚朴降低细胞迁移可能通过抑制ROS的产生。迁移指向性显示在图2G。在EF的存在只，肺癌细胞呈现朝向阳极（负的值）突出定向迁移。加入不同浓度的厚朴后，在迁移指向性略有下降为所有EF刺激的领域。 <img alt="图1" src="/files/ftp_upload/54397/54397fig1.jpg" /> 图1:化学剪切应力（CSS）芯片用于研究化学品的影响及剪切对人肺癌细胞强调2。3 （A）中的PMMA基板的三个层通过两个双面胶带结合在一起形成CSS的芯片。 （B）中集成的CSS芯片。 （C）上图:荧光和1-5细胞后CL的亮场图像与不同浓度的H 2 O 2处理。比例尺= 50微米。底部:平均荧光强度用SEM绘制在不同浓度的H 2 O 2。 （D）上图:在不同的剪切应力1-5细胞CL的荧光图像。比例尺= 50微米。底部:平均荧光强度用SEM绘制在不同的剪切应力。 （E）的顶部:CL的荧光和亮场图像与不同浓度的α生育酚的剪切应力被刺激后1-5细胞。比例尺= 50微米。底部:平均荧光强度用SEM绘制在不同浓度的α生育酚。数据这里介绍W¯¯最初发表于23参考。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54397/54397fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54397/54397fig2.jpg" /> 图2:化学电场（CEF）芯片用于研究化学品对人肺癌细胞22电场效应和。 聚甲基丙烯酸甲酯的（A）的一个层通过双面胶带结合到培养皿以形成CEF芯片。 （B）中的CSS芯片的流体的图案。 （C）的微流体芯片的等效电路。 （D）左:1-5细胞后CL的荧光和亮场图像与EF的不同强度的治疗。比例尺= 50微米。右:平均荧光用SEM强度绘制在EF的不同优势。 （E）左:荧光灯和1-5细胞后CL的亮场图像与联合厚朴酚和EFS治疗。比例尺= 50微米。右:平均荧光强度与SEM绘制在联合厚朴酚和EFS。 （F）的正与EF文件只（标记为对照）和合并的厚朴酚和EFS处理后CL1-5细胞的迁移速率（标示为和厚朴酚）。 （G）的正与EF文件只（标记为对照）和合并的厚朴酚和EFS处理后CL1-5细胞迁移指向性（标记为厚朴酚）。这里介绍的数据最初发表于22参考。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54397/54397fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli at JoVE.com

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

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 ...

designing-microfluidic-devices-for-studying-cellular-responses-under

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K 次观看.  National Taiwan University. 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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

生物学

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

A Microfluidic Technique to Probe Cell Deformability

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 efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

在定义良好的剪切应力下单细胞电电流的电生理记录

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...