该研究是由卫生部资助号R01NS050452和R01EB009429研究所。在外部进行悬臂芯片制造由合作者在位于康奈尔大学的纳米加工设施。在悬臂的制造过程中使用的所有设备被设在该设施。特别感谢小敏的Esch和Jean-马修普罗特其悬臂微加工的援助。由查尔斯·休斯，阿莱克斯Zelenin和埃里克·御从合成现实实验室在UCF的生成影视动画的悬臂功能。

1，悬臂式芯片制造在图1中提供了所描述的制造步骤示出的细节。 <ol><li>放置上的硅绝缘体（SOI）晶片在烘箱中烘在125℃下进行20分钟至脱水它们。 </li><li>淀积1.5微米厚的氧化硅层上，用等离子的脱水SOI晶片的把手层增​​强的化学气相沉积（PECVD）的工具。 </li><li>放置在旋涂机卡盘与装置层朝上的晶片。确保晶片的中心，取2毫升P20引物上的中心晶片，并旋转以3,000 rpm离心60秒以1000转数/秒的加速度。 P20引物促进光致抗蚀剂的器件层的密合性。 </li><li>而晶片仍然在旋涂机夹头，分配2毫升S1818光致抗蚀剂，在晶片的中心，和旋转的转数3000rpm，60秒，在1000转/秒的加速度与邻btain 1.5微米厚的光刻胶层。 </li><li>放置在晶片上的热板上，并持续1分钟进行软烤115℃。 </li><li>为了从悬臂掩模将图案转移到光致抗蚀剂的器件层上执行使用光刻接触掩模对准器的硬接触UV曝光。计算的基础上的125毫焦/厘米2的S1818光致抗蚀剂的曝光能量值的曝光时间。 </li><li>浸在726 MIF光刻胶显影剂晶片1分钟同时轻轻摇动晶片来回开发的光致抗蚀剂。 </li><li>用去离子水（DI）晶片与擦干，最好使用旋转清洗机（SRD）的工具。 </li><li>除去从晶片的边缘的光致抗蚀剂（最多5毫米的边缘）用棉棒蘸丙酮。 </li><li>装入晶片的深反应离子刻蚀（DRIE）工具，具有光致抗蚀剂面朝上，并运行一个配方通过德维蚀刻图案化硅层策层。埋入氧化层用作蚀刻停止，所以，用50％进行蚀刻，以100％以上的周期比预计是必要的，以确保完整的蚀刻。 </li><li>放置在热致抗蚀剂浴溶液晶片20分钟，剥去光致抗蚀剂。晶圆转移到快速转储冲洗机（QDR）冲洗用去离子水晶圆。以下的光刻胶剥离，加载晶片在SRD中的工具来冲洗和干燥晶片。检查在显微镜下的晶片，以确保如期悬臂模式出现。 </li><li>沉积1微米厚的一层未掺杂的氧化硅的利用PECVD工具在晶片的器件层上。该氧化层用作保护层的过程中进一步的处理步骤的悬臂。 </li><li>放置在旋涂机夹头朝上把手层晶片以开始晶片的背面侧的处理。确保晶片的中心，取2毫升P20引物上的晶片的中心，并旋以3000rpm为60秒，在1000转/秒的加速度。 </li><li>而晶片仍然在旋涂机夹头，分配2毫升SPR220-4.5光致抗蚀剂上的中心晶片，并旋转以2000rpm，持续45秒，以1000转数/秒的加速度以得到6.5微米厚的一层光致抗蚀剂。 </li><li>放置在接近热板的晶片进行软烘烤，在115℃进行2分钟。 </li><li>使用光刻接触对准器能够正面/背面对准，对准背侧窗口掩模的前侧悬臂模式和以从背面窗口掩模的图案转移到光致抗蚀剂的操作层上执行硬接触UV曝光。使用基于480毫焦/厘米2为SPR220-4.5光致抗蚀剂的曝光能量值计算的曝光时间。 </li><li>紫外曝光后，让晶片的下一个处理步骤之前，保持在一个暗区30分钟。 </li><li>通过浸入晶片开发光致抗蚀剂à726 MIF光刻胶显影剂2分钟，同时轻轻摇晃晶圆来回。 </li><li>用去离子水（DI）晶片与擦干，最好使用旋转清洗机（SRD）的工具。 </li><li>放置在烘箱中的晶片在90℃下12小时。 </li><li>刻蚀使用的RIE系统与氟化气体和配方用于蚀刻氧化物的晶片的背面氧化硅层。在晶片的背面上的图案化的氧化物作为蚀刻掩模，以便进一步处理。检查在显微镜下的晶片，以保证硅氧化物在曝光窗被完全蚀刻。 </li><li>除去用干净的房间拭子蘸取丙酮在晶片的边缘的光致抗蚀剂（最多5毫米的边缘）。 </li><li>加载在DRIE工具与晶片并运行一个配方的图案化操作层蚀刻至500μm的深度与埋入氧化层用作蚀刻停止。分割该蚀刻成多个运行，以防止过度加热晶片，这将导致硅的不一致的蚀刻。该蚀刻步骤完全消除悬臂下方的硅。 </li><li>执行使用25％稀释的HF蚀刻剂，剥离低于悬臂埋入氧化层和上悬臂的顶部的保护性氧化层的湿法蚀刻步骤。 注意:此步骤发行的硅悬臂梁的底面，也打开了一个窗口下方，以提供一个接入探测与激光的悬臂。 </li><li>冲洗用一系列的去离子水浴场，仔细用氮气干燥晶圆。由于悬臂是在他们的基地只支持，不直接对悬臂使用去离子水或惰性气体有力的喷雾剂。 </li><li>从沿着在手柄层的蚀刻步骤中产生的解理线晶片劈开单个芯片。 </li></ol> 2，细胞培养<ol><li>根据此前公布的方法17准备13F盖玻片。 注:如果13F海湾rslips不可用，可以使用任何疏水性表面与上述95°的接触角。 </li><li>消毒悬臂芯片和13F盖玻片的70％乙醇溶液风干的流罩。 </li><li>广场上的13层顶部盖玻片个人悬臂芯片标准的12孔板内。 </li><li>涂层与该生物聚合物或表面改性的细胞类型优化的悬臂根据标准细胞培养协议被使用。 </li><li>在其特定的生长培养基中重悬浮细胞至所需浓度。 注意:此协议将导致细胞脱落，通过悬臂窗口和未附着到所需悬臂表面有相当多的。细胞制剂因此，应当作出3-4倍比标准盖玻片制剂更加集中。例如，大鼠骨骼肌卫星细胞的播种典型地采用500至700个细胞/平方毫米15,16的接种密度，并以2,000个细胞/平方毫米使用悬臂衬底。优化实验，应进行新的细胞来源，以确定一个合适的接种密度。 </li><li>吸取200μl的细胞悬浮液上的悬臂晶片表面，确保介质的泡沫覆盖悬臂窗户完全。如果介质灯芯通过窗口，并不构成对悬臂顶部上的静电气泡取代13F盖玻片并重新尝试电镀。 </li><li>传送含有芯片的培养箱板和使细胞附着至少1小时（优选2-3小时）。 </li><li>在此之后的电镀周期，用无菌镊子芯片1毫升生长培养基转移到一个干净的好，没有一个13F盖玻片，加满油的文化。 </li><li>返回板的孵化器。 </li><li>根据他们的标准协议，用于在体外维持在盖玻片上维持细胞。对骨骼肌细胞，介质组合物的一个开关来诱导肌管形成一次细胞汇合将是必要的。 </li></ol> 3，安装硬件和软件的悬臂挠度分析<ol><li>将加热的培养皿成一个堂堂正正的电镜的阶段。 </li><li>添加3毫升目前的细胞悬浮在（+的10mM HEPES）馈送介质的加热显微镜阶段。 </li><li>安装在加热的培养皿的内侧不锈钢电极以15mm的间隔距离，并将它们连接到脉冲发生器，能够产生不同的强度，频率和波形的场刺激脉冲的，以允许系统以产生场刺激当适当的细胞。 </li><li>螺栓氦 - 氖激光器，装在XY平移阶段，在显微镜台的下侧，并调整它使得激光束以30°角relativ通过加热培养皿的底部导向E至悬臂的平面。 </li><li>螺栓象限光检测模块，安装在XY平移阶段，对显微镜载物台的下侧，并调整位置，以使反射的激光束的土地中的4个象限的中心， 图2提供了硬件设置的概况必要为实现所描述的协议。 </li><li>编写一个软件程序来控制线性致动器的跨悬臂扫描。写，参照图3中提供的流程图，该软件程序。编程为与该系统一起使用的图形界面，在图4中提供。 </li></ol> 4，录音悬臂式弯沉数据<ol><li>打开悬臂分析硬件和相关的软件。 </li><li>将加热阶段的热敏电阻到培养基中，然后等待它来读取37℃。 </li><li>插入悬臂芯片与cantileve阶段RS面向舞台的右侧。 </li><li>打开显微镜光源。 </li><li>聚焦显微镜使悬臂的边缘进入视野，并使用激光/光电探测器的控制软件来定位激光束对悬臂1。注意的提示:假设悬臂是面向舞台的右边，悬臂1是一个位于阵列的左上角和数字中的左下向下运行至16。悬臂17是在右上位置，并运行到32中的右下角（ 图5A）。 </li><li>按"PLAY"的录音软件。 </li><li>定位在光检测器，使得在x和y帧中的信号读出"0"，通过调节步进电机控制的光检测器。 </li><li>在激光/光检测器的控制软件中设置悬臂1的位置（ 图4）。 </li><li>移动激光到悬臂16的前端，则重复步骤4.7，并设置cantilevER在激光/光电探测器的控制软件16位。 </li><li>移动激光到悬臂32的前端，则重复步骤4.7，并设置悬臂32的位置，在激光/光检测器的控制软件。 </li><li>移动激光到悬臂17的前端，则重复步骤4.7，并设置悬臂17的位置，在激光/光检测器的控制软件。 </li><li>关掉显微镜光源与架空光在实验室。 </li><li>在刻录软件，按"记录"。 </li><li>设置脉冲发生器硬件到40毫秒，5 V脉冲1Hz的频率，并打开机器。 注意:任选地，采用优化的刺激方案为特定的细胞来源，在这一点上，所述设置是基于使用人类和啮齿动物细胞来源12,13,15,16收集到的数据的指导方针。 </li><li>使用激光/光电探测器的控制软件，设置跨越32悬臂阵列扫描硬件，在旺火上停5秒ê。 </li><li>当32悬臂的扫描完成后，关闭刺激器，然后停止录音软件，实现了数据文件。 </li><li>检查记录的跟踪每个悬臂的收缩活动的证据。请记下每个悬臂阳性反应。甲收缩被定义为峰，如果偏转是至少0.1 V以上的基线。 </li><li>从激光/光电探测器的控制软件的扫描协议，删除所有非敏感的悬臂。 </li><li>活动的悬臂可以再无刺激，以获得细胞的自发收缩活动的阅读来重新扫描。 </li><li>运行扫描具有或不具有广泛的领域的电刺激之后，另外的治疗性化合物的培养基中，以观察在培养的细胞中的功能性输出的效果。 </li><li>进行疲劳评估通过电刺激的细胞长时间和对照的水平扫描<html> actility测量需要多长时间的峰值力下降到低于特定阈值。 </li><li>在运动神经元中被保持在共培养与肌肉的实验中，测量经过治疗运动神经元，肌管悬臂共培养的神经肌肉接头处形成一个神经兴奋剂（如谷氨酸）或突触抑制剂（ 例如 ，D-筒箭毒碱）和扫描分别为16自发活动的增加和减少。 </li><li>作为执行决定的计划实验的需要具体的扫描。 </li></ol>悬臂挠度数据的5分析<ol><li>使用改性斯通方程15（下面详述），以生悬臂偏转的数据（以伏特）转换成应力在细胞层或肌管（帕斯卡），并直接测量细胞收缩力（在纳米牛顿）的读出: 广告/ 51866 / 51866eq1.jpg"/&gt;公式1 <img alt="式（2）" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" />式（2） </li><li>其中δ=悬臂尖端偏转和应力的肌管产生，σC =应力由肌管产生，假设一个均匀的厚薄膜的悬臂，C 检测器的整个宽度=照片与电压到激光位置的系统特定的系数-detector，θ=相对于悬臂的面内的激光和检测器的角度，E Si为硅的弹性模量， 吨Si为厚度悬臂的， 吨 f =肌管厚度，V = 硅的毒害的比率硅，L =悬臂长度，激光P =路径长度悬臂针尖至探测器和 w <sub&gt; Si为悬臂的宽度。的示意性说明在这些方程中使用的术语是在图6中提供。 </li><li>假设肌管是均匀的膜，在肌管的力等于在薄膜上的力，从而导致式（3）通过等同的力从应力的计算和假设小区的横截面面积被用于斯通的的应用程序，它方程。 <img alt="式（3）" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" />式（3） </li><li>以下功能的数据采集，固定悬臂芯片的免疫细胞化学分析或利用细胞使用标准技术的蛋白质或DNA分析。 </li><li>可选地，返回，以便在稍后的时间点，重新评估功能表现的细胞培养箱代替制备用于分子分析。 </li></ol>

<ol><li>Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+liberation+of+myogenic+cells+from+adult+rat+muscle%5BTitle%5D)+AND+%22Anat.+Rec%22%5BJournal%5D)">Enzymatic liberation of myogenic cells from adult rat muscle.</a> Anat. Rec. 180, 645-661 (1974).</li>
<li>Yasin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+quantitative+technique+for+growing+human+adult+skeletal+muscle+in+culture+starting+from+mononucleated+cells%5BTitle%5D)+AND+%22J.+Neurol.+Sci%22%5BJournal%5D)">A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells.</a> J. Neurol. Sci. 32, 347-360 (1977).</li>
<li>Dennis, R. G., Kosnik, P. E., 2nd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitability+and+isometric+contractile+properties+of+mammalian+skeletal+muscle+constructs+engineered+in+vitro%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev.+Biol.+Anim%22%5BJournal%5D)">Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro.</a> In Vitro Cell Dev. Biol. Anim. 36, 327-335 (2000).</li>
<li>Khodabukus, A., Baar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defined+electrical+stimulation+emphasizing+excitability+for+the+development+and+testing+of+engineered+skeletal+muscle%5BTitle%5D)+AND+%22Tissue+Eng+Part+C+Methods%22%5BJournal%5D)">Defined electrical stimulation emphasizing excitability for the development and testing of engineered skeletal muscle.</a> Tissue Eng Part C Methods. 18, 349-357 (2012).</li>
<li>Langelaan, M. L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+maturation+by+electrical+stimulation%3A+Differences+in+response+between+C2C12+and+primary+muscle+progenitor+cells%5BTitle%5D)+AND+%22Journal+of+tissue+engineering+and+regenerative+medicine%22%5BJournal%5D)">Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells.</a> Journal of tissue engineering and regenerative medicine. 5, 529-539 (2011).</li>
<li>Stephenson, G. M., O'Callaghan, A., Stephenson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-fiber+study+of+contractile+and+biochemical+properties+of+skeletal+muscles+in+streptozotocin-induced+diabetic+rats%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.</a> Diabetes. 43, 622-628 (1994).</li>
<li>Harber, M., Trappe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+muscle+fiber+contractile+properties+of+young+competitive+distance+runners%5BTitle%5D)+AND+%22Journal+of+applied+physiology+%28Bethesda%2C+Md%3A+1985%29%22%5BJournal%5D)">Single muscle fiber contractile properties of young competitive distance runners.</a> Journal of applied physiology (Bethesda, Md: 1985). 105, 629-636 (2008).</li>
<li>Hvid, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+days+of+muscle+disuse+impairs+single+fiber+contractile+function+in+young+and+old+healthy+men%5BTitle%5D)+AND+%22Experimental+gerontology%22%5BJournal%5D)">Four days of muscle disuse impairs single fiber contractile function in young and old healthy men.</a> Experimental gerontology. 48, 154-161 (2013).</li>
<li>Edman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contractile+performance+of+striated+muscle%5BTitle%5D)+AND+%22Advances+in+experimental+medicine+and+biology%22%5BJournal%5D)">Contractile performance of striated muscle.</a> Advances in experimental medicine and biology. 682, 7-40 (2010).</li>
<li>Krivickas, L. S., Walsh, R., Amato, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+muscle+fiber+contractile+properties+in+adults+with+muscular+dystrophy+treated+with+MYO-029%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029.</a> Muscle Nerve. 39, 3-9 (2009).</li>
<li>Sung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfabricated+mammalian+organ+systems+and+their+integration+into+models+of+whole+animals+and+humans%5BTitle%5D)+AND+%22Lab+on+a+chip%22%5BJournal%5D)">Microfabricated mammalian organ systems and their integration into models of whole animals and humans.</a> Lab on a chip. 13, 1201-1212 (2013).</li>
<li>Wilson, K., Molnar, P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+functional+myotubes+with+a+Bio-MEMS+device+for+non-invasive+interrogation%5BTitle%5D)+AND+%22Lab+on+a+chip%22%5BJournal%5D)">Integration of functional myotubes with a Bio-MEMS device for non-invasive interrogation.</a> Lab on a chip. 7, 920-922 (2007).</li>
<li>Wilson, K., Das, M., Wahl, K. J., Colton, R. J., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+contractile+stress+generated+by+cultured+rat+muscle+on+silicon+cantilevers+for+toxin+detection+and+muscle+performance+enhancement%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement.</a> PLoS ONE. 5, (2010).</li>
<li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+Force+Microscope%5BTitle%5D)+AND+%22Physical+Review+Letters%22%5BJournal%5D)">Atomic Force Microscope.</a> Physical Review Letters. 56, 930-933 (1986).</li>
<li>Pirozzi, K. L., Long, C. J., McAleer, C. W., Smith, A. S., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlation+of+embryonic+skeletal+muscle+myotube+physical+characteristics+with+contractile+force+generation+on+an+atomic+force+microscope-based+bio-microelectromechanical+systems+device%5BTitle%5D)+AND+%22Applied+physics+letters%22%5BJournal%5D)">Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.</a> Applied physics letters. 103, 83108(2013).</li>
<li>Smith, A., Long, C., Pirozzi, K., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+functional+system+for+high-content+screening+of+neuromuscular+junctions+in+vitro%5BTitle%5D)+AND+%22Technology%22%5BJournal%5D)">A functional system for high-content screening of neuromuscular junctions in vitro.</a> Technology. 1, 37-48 (2013).</li>
<li>Stenger, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes%3A+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth.</a> Journal of the American Chemical Society. 114, 8435-8442 (1992).</li>
<li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">A defined long-term in vitro tissue engineered model of neuromuscular junctions.</a> Biomaterials. 31, 4880-4888 (2010).</li>
<li>Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc%3A+Sensory+neuron+innervation+of+intrafusal+muscle+fibers%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li>
<li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+tissue+engineering%3A+A+maturation+model+promoting+long-term+survival+of+myotubes%2C+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li>
<li>Das, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev+Biol+Anim%22%5BJournal%5D)">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li>
<li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture%5BTitle%5D)+AND+%22Brain+research%22%5BJournal%5D)">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li>
<li>Hickman, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry%5BTitle%5D)+AND+%22Journal+of+Vacuum+Scienc.%2C+%26+Technology+A%3A+Vacuum%2C+Surfaces%2C+and+Films%22%5BJournal%5D)">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., & Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li>
<li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system%5BTitle%5D)+AND+%22Biotechnology+progress%22%5BJournal%5D)">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li>
<li>Rumsey, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Node+of+Ranvier+formation+on+motor+neurons+in+vitro%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li>
<li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior%5BTitle%5D)+AND+%22International+journal+of+biomedical+engineering+and+technology%22%5BJournal%5D)">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li>
<li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+human+fetal+spinal+cord+stem+cell+line%2C+NSI-566RSC%2C+and+its+induction+to+functional+motoneurons%5BTitle%5D)+AND+%22Journal+of+Tissue+Engineering+and+Regenerative+Medicine%22%5BJournal%5D)">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li>
<li>Varghese, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643(2010).</li>
<li>Natarajan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional%2C+high+information+content+drug+screening+platform%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li>
<li>Davis, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture%3A+An+Axon-Oligodendrocyte+Interaction+Model%5BTitle%5D)+AND+%22Journal+of+biomaterials+and+tissue%22%5BJournal%5D)">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li>
<li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+In+Vitro+Feed-Forward+Networks%5BTitle%5D)+AND+%22J+Biotechnol+Biomater%22%5BJournal%5D)">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2(2013).</li>
</ol>

The authors have nothing to disclose.

在分析微尺度悬臂蜂窝收缩证据的关键步骤是在放置于显微镜载物台的悬臂芯片，并且激光和光检测器的阵列中的角悬臂的末端的随后对准。如果不正确地进行，则该软件将无法推断剩余的悬臂的位置的阵列中，从而可能导致假阴性积累数据收集期间。运营商应注意，以确保悬臂芯片校准激光的位置之前，躺在平齐培养皿的底部。如果芯片坐在在盘的角度，它会改变反射激光束的路径和混淆的数?...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table><tbody><tr><td>Primary rat muscle growth medium</td><td></td><td></td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B27 (50x)</td><td>Life Technologies</td><td>17504044</td><td>1x</td></tr><tr><td>Glutamax (100x)</td><td>Life Technologies</td><td>35050061</td><td>1x</td></tr><tr><td>G5 supplement</td><td>Life Technologies</td><td>17503-012</td><td>1x</td></tr><tr><td>Glial-Derived Neurotrophic Factor</td><td>Cell sciences</td><td>CRG400B</td><td>20 ng/ml</td></tr><tr><td>Brain-Derived Neurotrophic Factor</td><td>Cell sciences</td><td>CRB600B</td><td>20 ng/ml</td></tr><tr><td>Ciliary Neurotrophic Factor</td><td>Cell sciences</td><td>CRC400A</td><td>40 ng/ml</td></tr><tr><td>Neurotrophin-3</td><td>Cell sciences</td><td>CRN500B</td><td>20 ng/ml</td></tr><tr><td>Neurotrophin-4</td><td>Cell sciences</td><td>CRN501B</td><td>20 ng/ml</td></tr><tr><td>Acidic Fibroblast Growth Factor</td><td>Life Technologies</td><td>13241-013&amp;nbsp;</td><td>25 ng/ml</td></tr><tr><td>Cardiotrophin-1</td><td>Cell sciences</td><td>CRC700B</td><td>20 ng/ml</td></tr><tr><td>Heparin Sulphate</td><td>Sigma</td><td>D9809&amp;nbsp;</td><td>100 ng/ml</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Sigma</td><td>L5158&amp;nbsp;</td><td>20 ng/ml</td></tr><tr><td>Vitronectin</td><td>Sigma</td><td>V0132</td><td>100 ng/ml</td></tr><tr><td>Primary rat muscle differentiation medium</td><td></td><td></td><td></td></tr><tr><td>NB Activ 4</td><td>Brain Bits LLC</td><td>NB4-500</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Class 2 red diode laser</td><td>Newport</td><td></td><td></td></tr><tr><td>Photo-detector</td><td>Noah Industries</td><td></td><td></td></tr><tr><td>Model 2100 Pulse stimulator</td><td>A-M systems</td><td></td><td></td></tr><tr><td>Multiclamp 700B Digitizer</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Patch clamp microscope and stage</td><td>Olympus</td><td></td><td></td></tr><tr><td>Delta T4 culture dish controller</td><td>Bioptechs</td><td></td><td></td></tr><tr><td>Axoscope software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>LabVIEW software</td><td>National Instruments</td><td></td><td></td></tr><tr><td>37&lt;sup&gt; o&lt;/sup&gt;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>NAPCO</td><td></td><td></td></tr><tr><td>Class 2 microbiological flow hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Pipettes and tips</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

的多个预测和生物相关的体外测定法的发展的前提是多功能的细胞培养系统，促进种子细胞的功能评价的进步。为此，微型悬臂技术提供了一个平台，用以测量各种细胞类型，包括骨骼，心脏和平滑肌细胞的收缩的功能，通过收缩引起的基板弯曲的评估。复用的悬臂阵列的应用提供了一种方式来开发中等至高通量的协议，以评估药物疗效和毒性，疾病的表型和进展，以及神经肌肉和其他细胞 - 细胞相互作用。该原稿提供的信息用于制造可靠的悬臂阵列用于此目的，并且需要在这些表面上成功地培养细胞的方法。设置在需要进行官能肛门的步骤进一步描述收缩的细胞类型ysis保持上使用一种新的激光和光检测系统的这种阵列。到这样的技术可以应用于提供亮点的精度和收缩功能可以利用本系统的分析的可再现性的有代表性的数据，以及在广泛的研究。成功的广泛采用该系统的能提供研究者的手段来在体外进行快速，低成本的功能性研究，导致了组织的性能，疾病发展和响应于新的治疗治疗更准确的预测。

收缩细胞对悬臂成功的文化是一个相对简单的过程，利用标准的细胞培养技术（ 图5）。悬臂支撑承包细胞的百分比将取决于细胞类型而异正在研究和具体的栽培技术采用。使用从大鼠后肢来源的原胚细胞，收缩活性，对悬臂检查的12％检测到的组（n = 4）。使用所描述的激光和光检测器系统的收缩功能的分析提供了关于所接种的细胞的功能的成熟准确的实时数据。使用标准的电生理?...

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

这个协议描述了用于测量肌肉细胞的收缩力在体外使用的微型硅悬臂梁的柔韧培养表面。细胞收缩引起悬臂弯曲，可以测量，记录和转换成力读数，用于测量收缩功能的体外提供一种非侵入性和可扩展的系统。

微型硅悬臂梁的利用率，评估细胞收缩功能<em&gt;在体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

utilization-microscale-silicon-cantilevers-to-assess-cellular

Research

JoVE Journal

Bioengineering

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

10.2K 次观看.  University of Central Florida. 这个协议描述了用于测量肌肉细胞的收缩力在体外使用的微型硅悬臂梁的柔韧培养表面。细胞收缩引起悬臂弯曲，可以测量，记录和转换成力读数，用于测量收缩功能的体外提供一种非侵入性和可扩展的系统。 

视频: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

科研

生物工程

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工程学

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

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

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

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

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

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

使用微图案弹性体对单细胞收缩力进行简化的高通量分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

机械节点孔传感:用于多参数单细胞粘弹性测量的快速、无标记平台

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

用于评估细胞运动和相关信号事件的机械控制的单细胞Durotaxis测定

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

生物学

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

微型硅悬臂梁的利用率，评估细胞收缩功能

摘要

探索更多视频