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>
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F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+embryonic+skeletal+muscle+myotube+physical+characteristics+with+contractile+force+generation+on+an+atomic+force+microscope-based+bio-microelectromechanical+systems+device.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+system+for+high-content+screening+of+neuromuscular+junctions+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes:+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">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 border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</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

10.1K 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

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

科研

生物工程

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

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

由微型条纹分析技术在微流控设备混合不均匀

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

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

测量与建模干燥收缩人类角质层

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.