作者谨感谢那个技术学院联邦快递在孟菲斯大学的支持。作者要感谢学生在机械工程系高级设计项目组在孟菲斯（大卫·巴特勒，卡特成龙，多米尼克骑士，雅各布谢弗）的大学，丹尼尔·科恩来自孟菲斯大学的工程技术部为电机控制和腾斌和Charlean Luellen女士的细胞培养博士的帮助。这项工作是由K01 HL120912（ER）和R01 HL123540（CMW）的支持。

1.构造膜与井壁为细胞保留培养基（参见图1D为最终产品） <ol><li>使用涂覆有胶原I聚二甲基硅氧烷（PDMS）片，切开柔性膜的轮廓用手术刀或模具。 </li><li>将每个膜在60毫米的培养皿进行存储。 </li><li>创建墙: <ol><li> PDMS混合在10:弹性体A的1重量比为弹性体B（固化剂）。 </li><li>倒入加入5ml充分混合成PDMS 50ml试管。 </li><li>放置50ml试管用未固化的PDMS水平在杂交烘箱。 </li><li>在固化时使用转子函数来涂覆管的内壁8的转速。在RT，PDMS的将完全固化2天。 </li><li>在无菌细胞培养罩除去从管中的PDMS。 </li><li>使用一个新的刀片，分区的PDMS的气缸段4毫米的高度。 </li><li>放置的部分，这将作为膜わLLS，成不使一个培养皿容器壁变得折痕。 </li></ol></li><li>在一个膜选择和中心一面墙上的PDMS同时保持两个培养皿。 </li><li>轻轻地放在未固化的PDMS:在墙壁上的外周（10比1的比例），以用作胶水。防止形成的壁和所述膜之间的间隙，因为它的存在，以保持液体。 </li><li>把这个包含完成膜和覆盖在烘箱中于70℃下放置24小时以固化各培养皿。 </li></ol> 2.相关电机转动圈与夹具位移或径向生长的校准（图2） <ol><li>启动软件控制电动机。 </li><li>置换设备的使用电机软件手动设置的夹子。 </li><li>测量距离，并记录在最小和最大的夹具位移相对夹钳之间的电动机计数位置。 </li><li>计算在对d变化百分比夹具作为电动机计数（〜0-75k计数）的功能之间istance。这将表明实现了对膜的最大潜在应变。 </li></ol>拉伸对小鼠肺上皮细胞3.应用（MLE12） <ol><li>种子MLE12细胞以每孔250万细胞上的柔性膜（步骤1）将在两天内汇合。接种密度可以变化为不同的细胞类型。 </li><li>确保机械致动器处于完全松弛位置。 <ol><li>除去细胞的培养基。 </li><li>使用1.5毫米活检穿孔，冲两个孔在每六个夹具（参见图1D）膜的标签。孔的径向位置决定预张力的量的膜经历最初。冲孔在20.5毫米的膜片的半径，如果没有预张力是理想的。 </li><li>定位膜上与冲压孔排队有夹具内引脚的担架。将顶部夹具到位。拧紧螺钉一次双方交替。 </li><li>加入1毫升细胞培养基。 </li></ol></li><li>将设备放在显微镜阶段定心与所述光路的膜的中间。 </li><li>使用磁带或磁体（如果可能的话）将设备固定到舞台上。一旦固定，控制面内（平行于膜）的机械装置与显微镜的载物台控制的和垂直（z方向）。 </li><li>与由手动控制电动机旋转20的位置和速度由制造商提供的软件应用伸展。 </li></ol> 4.测量线粒体活性氧（ROS） <ol><li>一旦细胞汇合，加入1-2毫升RT DMEM中与线粒体超氧化物指示器（5μM终浓度）直接作用于细胞。 </li><li>孵育10分钟，在37℃。 </li><li>洗涤细胞轻轻三次缓冲在水浴温热至37℃。 </li><li>立即放置在担架上（步骤2.2）已经到位的柔性膜的直立共聚焦显微镜下观察。 </li><li>加入1毫升的DMEM与无酚红培养基和HEPES 25mM的。 </li><li>设置激发/发射过滤器五百八十〇分之五百十纳米。 </li><li>图片多个领域，每15分钟创造线粒体超氧生产所需的时间过程。 </li><li>从拍摄的图像，记录的荧光强度在每个时间间隔中利用软件能够量化荧光强度直方图。 </li></ol> 5.应用拉伸和原子力显微镜（AFM） 注:提供的特定原子力显微镜和光学显微镜的组合（ 图4和材料清单）这些步骤。 <ol><li>准备的AFM实验。 <ol><li>增加的原子力显微镜头的高度，以它的在z方向上的最大位置。 </li><li>把扩展在AFM的腿解除平面处的AFM悬臂接触样品。试样和原子力显微镜头需要被提升，以适应机械装置的高度。 </li></ol></li><li>准备光学显微镜（如果可用）。 <ol><li>取出AFM扫描板。删除了预期的目的。 </li><li>添加间隔物的目标。的隔离物的高度将取决于目标和具体的AFM设置，但它是必要的，如果光学成像期望因为观察面将是一个等于担架和适配器高度（ 图移在z方向图5A）。需要注意的是在原子力显微镜通常提供低倍率摄像从装置上方的光路。 </li><li>安装了预期的目的放回其位置。将扫描仪背面上的原子力显微镜。 </li><li>启动AFM软件。启动所有必需的光源包括光源的荧光测量。 </li><li>安装有一个悬臂梁的芯片适合于所需的测量。测定活细胞的弹性模量时200 PN / nm以下刚性是优选的。 </li><li>对准激光，并根据制造商对安装在该装置上的玻璃盖玻片建议校准悬臂刚度。 </li></ol></li><li>制备的膜作为第1步，但进行如下修改。 <ol><li>立即安装在膜上的拉伸装置之前，切割的壁至约1毫米的高度。这可以防止所经历的膜壁和负载元件之间的干扰。 </li><li>如上所述3.2.1-3.2.3安装膜的机械装置上。 </li><li>除去细胞的培养基，以防止损坏的原子力显微镜扫描仪中的情况下泄漏的。 </li><li>耦合装置与适配器（ 图5A）。将机械装置与扫描仪上的转接器。 </li><li>拉伸膜到期望的拉伸应变水平。 </li></ol></li><li&gt;纳米压痕拉伸细胞。 <ol><li>添加的媒体有限的（&lt;0.5毫升）卷上的细胞，以避免AFM扫描仪或显微镜损害，由于泄漏。 </li><li>接合悬臂梁与膜。 </li><li>按照特定的原子力显微镜装置的协议进行扫描感兴趣的领域。 </li></ol></li></ol>

<ol>
	<li>Tschumperlin, D. J., Boudreault, F., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+new+opportunities+in+lung+mechanobiology.">Recent advances and new opportunities in lung mechanobiology.</a> J Biomech. 43, 99-107 (2010).</li><li>Waters, C. M., Roan, E., Navajas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comprehensive Physiology. , (2011).</li><li>Majkut, S., Dingal, P. C. D. P., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+Sensitivity+and+Mechanotransduction+during+Heart+Development.">Stress Sensitivity and Mechanotransduction during Heart Development.</a> Current Biology. 24, R495-R501 (2014).</li><li>Hoffman, B. D., Grashoff, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+molecular+processes+mediate+cellular+mechanotransduction.">Dynamic molecular processes mediate cellular mechanotransduction.</a> Nature. 475, 316-323 (2011).</li><li>Wang, N., Butler, J. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+across+the+cell-surface+and+through+the+cytoskeleton.">Mechanotransduction across the cell-surface and through the cytoskeleton.</a> Science. 260, 1124-1127 (1993).</li><li>Liu, M., Tanswell, A. K., Post, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+force-induced+signal+transduction+in+lung+cells.">Mechanical force-induced signal transduction in lung cells.</a> Am J Physiol. 277, L667-L683 (1999).</li><li>Janmey, P. A., McCulloch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+integrating+cell+responses+to+mechanical+stimuli.">Cell mechanics: integrating cell responses to mechanical stimuli.</a> Annu Rev Biomed Eng. 9, 1-34 (2007).</li><li>Waters, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+mechanotransduction.">Reactive oxygen species in mechanotransduction.</a> Am J Physiol Lung Cell Mol Physiol. 287, L484-L485 (2004).</li><li>Chapman, K. E., 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=Cyclic+mechanical+strain+increases+reactive+oxygen+species+production+in+pulmonary+epithelial+cells.">Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 289, L834-L841 (2005).</li><li>Chu, E. K., Whitehead, T., Slutsky, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cyclic+opening+and+closing+at+low-+and+high-volume+ventilation+on+bronchoalveolar+lavage+cytokines.">Effects of cyclic opening and closing at low- and high-volume ventilation on bronchoalveolar lavage cytokines.</a> Crit Car Med. 32, 168-174 (2004).</li><li>Tschumperlin, D., Margulies, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equibiaxial+deformation-induced+injury+of+alveolar+epithelial+cells+in+vitro.">Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro.</a> Am J Physiol. 275, L1173-L1183 (1998).</li><li>Vlahakis, N. E., Hubmayr, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+failure+in+ventilator-injured+lungs.">Cellular stress failure in ventilator-injured lungs.</a> Am J Respir Crit Care Med. 171, 1328-1342 (2005).</li><li>Roan, E., 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=Hyperoxia+alters+the+mechanical+properties+of+alveolar+epithelial+cells.">Hyperoxia alters the mechanical properties of alveolar epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 302, L1235-L1241 (2012).</li><li>Gamerdinger, 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=Mechanical+load+and+mechanical+integrity+of+lung+cells+-+Experimental+mechanostimulation+of+epithelial+cell-+and+fibroblast-monolayers.">Mechanical load and mechanical integrity of lung cells - Experimental mechanostimulation of epithelial cell- and fibroblast-monolayers.</a> J Mech Behav Biomed Mater. 4, 201-209 (2014).</li><li>Hayashi, K., Naiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+remodeling+of+vascular+wall;+biomechanical+response+to+hypertension.">Adaptation and remodeling of vascular wall; biomechanical response to hypertension.</a> J Mech Behav Biomed Mater. 2, 3-19 (2009).</li><li>Villemure, I., Stokes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+plate+mechanics+and+mechanobiology.+A+survey+of+present+understanding.">Growth plate mechanics and mechanobiology. A survey of present understanding.</a> J Biomech. 42, 1793-1803 (2009).</li><li>Waters, C. 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=A+system+to+impose+prescribed+homogenous+strains+on+cultured+cells.">A system to impose prescribed homogenous strains on cultured cells.</a> J Appl Physiol (1985). 91, 1600-1610 (2001).</li><li>Gerstmair, A., Fois, G., Innerbichler, S., Dietl, P., Felder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+device+for+simultaneous+live+cell+imaging+during+uni-axial+mechanical+strain+or+compression.">A device for simultaneous live cell imaging during uni-axial mechanical strain or compression.</a> J Appl Physiol (1985). 107, 613-620 (1985).</li><li>Dassow, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+measure+mechanical+properties+of+pulmonary+epithelial+cell+layers.">A method to measure mechanical properties of pulmonary epithelial cell layers.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 101, 1164-1171 (2013).</li><li>Chapman, 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=Cyclic+mechanical+strain+increases+reactive+oxygen+species+production+in+pulmonary+epithelial+cells.">Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 289, L834-L841 (2005).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+stretch,+reactive+oxygen+species,+and+vascular+remodeling.">Cyclic stretch, reactive oxygen species, and vascular remodeling.</a> Antioxid Redox Signal. 11, 1651-1667 (2009).</li><li>Turrens, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+formation+of+reactive+oxygen+species.">Mitochondrial formation of reactive oxygen species.</a> J Physiol. 552, 335-344 (2003).</li><li>Wang, 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=Superoxide+flashes+in+single+mitochondria.">Superoxide flashes in single mitochondria.</a> Cell. 134, 279-290 (2008).</li><li>Pouvreau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+flashes+in+mouse+skeletal+muscle+are+produced+by+discrete+arrays+of+active+mitochondria+operating+coherently.">Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently.</a> PLoS One. 5, (2010).</li><li>Yalcin, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cytoskeletal+structure+and+mechanics+on+epithelial+cell+injury+during+cyclic+airway+reopening.">Influence of cytoskeletal structure and mechanics on epithelial cell injury during cyclic airway reopening.</a> Am J Physiol Lung Cell Mol Physiol. 297, L881-L891 (2009).</li><li>Jacob, A. M., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atelectrauma+disrupts+pulmonary+epithelial+barrier+integrity+and+alters+the+distribution+of+tight+junction+proteins+ZO-1+and+claudin+4.">Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4.</a> J Appl Physiol. 113, 1377-1387 (2012).</li><li>DiPaolo, B. C., Lenormand, G., Fredberg, J. J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+magnitude+and+frequency-dependent+actin+cytoskeleton+remodeling+in+alveolar+epithelia.">Stretch magnitude and frequency-dependent actin cytoskeleton remodeling in alveolar epithelia.</a> Am J Physiol Cell Physiol. 299, C345-C353 (2010).</li></ol>

作者宣称，他们有没有竞争的财务权益。

对于在机械拉伸活细胞成像的独特装置的开发;这个装置是用在协议研究肺上皮细胞的力学生物学。在初步研究中，人们发现，单个保持伸展刺激生产在支气管上皮细胞线粒体超氧化物。此外，证实了机械应变水平的增加造成直接损伤肺泡上皮细胞的一个单层的完整性。 进行这些初步实验中，该装置被第一校准，然后将其结果表明，在膜的应变被传递到细胞中（ 图2-3）。整体的设备执行良好，由图3E中所示的膜应变（用标记在膜上测定）和细胞株（由原子核之间的距离测量）之间的密切相关性所指示的。然而，有斯特拉之间观察到一些细微的差别在测量上的方法中的膜和标记的返回曲线， 如图2D所示 。这可能是由于在该装置后冲和可能与夹紧的问题。此外，需要一种通用的安装机构，用于该装置，使之能够有效地在显微镜用不同的配置中使用。这将减少，同时保持稳固的连接到显微镜的设备和图像采集的定位之间所花费的时间。眼儿的膜用于将次要预应变施加到膜，以防止形成的初始压缩状态由于夹紧弹性材料制成的。压缩状态也导致减少最大适用应变。 定制设备的设计，以适应在庇护MFP3D原子力显微镜，允许在机械拉伸纳米压痕活细胞。我们不知道的任何其它装置，其能够在在成像平面，可以在AFM中运作ducing各向同性应力状态。该设备也被设计为连续使用在细胞培养孵育箱（保持在37℃下在潮湿的，无菌环境中，用5％的CO 2）。以孵化器内运行的能力允许细胞经历长期舒展方案的研究。 有几个限制了该方法。首先，作为膜伸展，焦点的字段移出平面的，虽然最低限度（〜100微米）。这将成为一个限制通过拉伸方案追踪单元的场时，由于视场改变在膜膨胀的过程中。即使直径22 mm的内膜经历0.23-0.25应变，这内径体验更多变应变场以外的地区。虽然成像研究很容易限于此中心区域，有可能这些细胞响应于较高水平的应变的在外部区域可以影响细胞的中心区域内的响应。在感兴趣较大面积的均匀应变分布可以与改进的膜设计来实现。的PDMS膜是自体荧光的波长通常用于荧光指示剂，并且这限制了能力，用于从膜下成像荧光细胞。出于这个原因，一个堂堂正正的显微镜利用在我们ROS生产研究上述水浸泡目标。这对于研究中，原子力显微镜和荧光成像被组合的限制。 最常见的商业装置（Flexcell牵张FX 5000张力系统），用于将机械应变的细胞结合施加拉伸应变在膜上而移入和移出所述光平面的可控真空系统。该膜和夹具设计本文中所呈现创建双轴应变场，其中应变是各向同性的，并且仍然在平面allowin摹实时成像。稍作修改到膜的设计，担架能够产生单轴拉伸模式，以及。如果细胞首先接种在扩张膜，面内压缩载荷也可以应用。该装置能够达到生理和病理水平的应变。总之，新的设备和协议被开发，可用于施加机械应变的活细胞，可以使用荧光显微镜和原子力显微镜纳米压痕进行成像。

细胞进行机械载荷在许多组织中，而这种机械刺激已经显示出促进基因表达的变化，生长因子，细胞因子或细胞外基质的重塑释放的模式和细胞骨架1-4。从这样的机械刺激转导的胞内信号，以通过机械传导5-7的过程发生。在呼吸系统，机械传导的一个结果是增加的活性氧（ROS）的肺上皮细胞中的环状拉伸应变的存在下8,9-和促炎细胞因子10。强有力的证据也表明，过量的拉伸应变导致直接伤害到肺泡上皮中，除了元件11-14的生化反应。虽然这里的重点主要是对肺细胞的机械变形的响应，诱发力传导通路起到BAS关键作用在人体内许多组织，包括血管紧张15的调节和生长板16的发展的集成电路功能。 的在机械传导日益增长的兴趣已经导致许多装置开发用于生理学相关的机械负载的应用，培养细胞和组织。特别是，施加拉伸应变，这是机械负荷经历组织的常见形式，设备是受欢迎11,17-19。然而，许多现有的装置被设计或者作为生物反应器用于组织工程应用或不利于实时成像伸展。因此，有必要开发工具和方法，可以可视化的细胞和组织中的张力，以促进机械传导的途径的调查。 此处，面内机械拉伸装置被设计和协议被开发应用米到组织，同时允许在实时（ 图1A-D）的生物化学和机械响应的成像ultiple形式株和细胞。该装置利用布置在圆周抓住一个柔性膜和施加在平面内，径向膨高达约20％（ 图1B）6均匀间隔夹具。所述致动装置可放置在细胞培养孵化器在延长的时间周期，而电机（ 图1C）位于所述培养箱外并通过由电动机供应商所提供的专用软件控制。电动机被连接到线性驱动器，其旋转一个内凸轮，驱动六担架夹子均匀地拉伸和放松。 除了机械设备，定制柔性膜由市售细胞培养准备膜产生的机械系统中使用。然后圆形壁（具有直径约28毫米）作了并附着到柔性膜使细胞只能在良好描述应变轮廓的这个区域进行培养。为了确定所述致动装置内的放置这些膜是否会提供均匀的和各向同性的应变在弹性膜的中心，有限元分析，使用市售的软件（ 图1E-F）进行的。灵活的膜仿照对称的边界条件与利用网格的所有四边形单元。见于最大主应变的图1F所示的等高线图的同心圆环指示菌株的各向同性分布。 所经历的膜的应变是由通过负载（ 图2）的记录标记的图像进行测量。 图2D示出了在径向和轴向方向测得的平均膜应力为近似线性相对于所施加的电动机向上计数到20％的最大线性应变。有腹胀期间与回缩回到静止位置时相比，测量测得的应变水平之间没有差异显著。接着，人支气管上皮细胞（16HBE），核上的定制柔性膜培养的位移进行测定。在16HBE细胞的荧光标记的吲哚（DAPI）细胞核使用共聚焦显微镜下一个20X物镜成像，而全细胞位移的测定使用记录用数字显微镜相衬图像。正如在图3中 ，由核位移测量的应变是类似于由斑纹的位移对膜测得的，高达约20％的线性应变。这证实了施加到膜的应变被传递到贴壁细胞。描述在传统显微镜的使用自定义设备的协议和原子力microscope被在下面的步骤提供。

<table><tbody><tr><td>SmartMotor NEMA 34: 3400 Series</td><td>MOOG Animatics</td><td>SM3416D</td><td>Integrated motor, controller, amplifier, encoder and communications bus</td></tr><tr><td>Flexcell Membrane (Collagen I coated)</td><td>Flexcell International Corp</td><td>SM2-1010C</td><td>3.5&quot;&amp;nbsp;x&amp;nbsp;5.25&quot;&amp;nbsp;x&amp;nbsp;0.020&quot;</td></tr><tr><td>Sylgard 184</td><td>Dow Corning Corporation</td><td></td><td>10:1</td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H1399</td><td>DAPI stain</td></tr><tr><td>MitoSOX</td><td>Sigma-Aldrich</td><td>M36008</td><td></td></tr><tr><td>Tiron</td><td>Sigma-Aldrich</td><td>D7389&amp;nbsp;</td><td>mitochondrial superoxide label</td></tr><tr><td>DMEM</td><td></td><td></td><td>superoxide inhibitor</td></tr><tr><td>FBS</td><td></td><td></td><td></td></tr><tr><td>HEPES</td><td></td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>Fisher Scientific</td><td>06-443-19</td><td>Any centriguge tube can be used to create an area for imaging.</td></tr><tr><td>Hybridization oven</td><td>Bellco Glass</td><td></td><td></td></tr><tr><td>MLE12 Cells</td><td>ATCC</td><td>CRL-2110</td><td>Mouse Lung Epithelial Cells&amp;nbsp;</td></tr><tr><td>16HBE cells</td><td>ATCC</td><td>CRL-2741</td><td>Human Bronchial Epithelial Cells</td></tr><tr><td colspan="4">&lt;strong&gt;AFM Indentation Experiments&lt;/strong&gt;</td></tr><tr><td>Cantilever Beams for Nano-indentation</td><td>Budget Sensors</td><td>Si-Ni30</td><td></td></tr><tr><td>AFM&amp;nbsp;</td><td>Asylum Research</td><td>MFP3D</td><td></td></tr><tr><td>Olympus microscope</td><td>Olympus</td><td>IX-71</td><td>Inverted microscope with 20X and 40X objectives.</td></tr><tr><td>AFM Leg Extenders</td><td>Asylum Research</td><td>Not available</td><td>AFM microscope</td></tr><tr><td colspan="4">&lt;strong&gt;Finite Element Analyses&lt;/strong&gt;</td></tr><tr><td>ABAQUS</td><td>Simulia</td><td>6.12</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Software&lt;/strong&gt;</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Microscopes&lt;/strong&gt;</td></tr><tr><td>Digital microscope</td><td>Life Technologies</td><td>EVOS XL Core</td><td>Initially a self standing company, now owned by Life Technologies.</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 710</td><td>2-photon upright microscope</td></tr></tbody></table>

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.

活性氧和变形 先前的研究已经响应于循环拉伸21中所示的增加的活性氧（ROS）的气道和肺泡上皮细胞。活性氧包括分子和来自于分子氧具有高反应性的脂质，蛋白质，多糖和核酸22-24自由基。 ROS用作共用的细胞内信号来调节离子通道功能，蛋白激酶/磷酸酶的激活和基因表达，但过量的或未调节的产生可以向凋亡和坏死性细胞死亡，神经变性，动脉粥样硬化，糖尿病和癌症24,25。超氧化物，预光标为其他形式的ROS，可以制造作为线粒体呼吸的副产物，当电子从电子传递链泄漏。以往的研究表明，周期性牵张刺激了生产超氧化物通过NADPH氧化酶系统（烟酰胺腺嘌呤二核苷酸磷酸氧化酶）和线粒体电子传递链（复合物I和III）的组合。已经提出了这种刺激是由于线粒体21可能直接失真由于连接到细胞骨架。为了检验这一假设，细胞拉伸装置的开发，使我们可以在ROS产生于活细胞响应机械拉伸影像学改变。在这里，由因拉伸支气管上皮细胞产生的线粒体活性氧使用自定义设备和协议进行测量。在没有机械拉伸的，ROS产生没有显著在60分钟（ 图4）增加。当单个的17％拉伸施加和维持，有所增加线粒体ROS该持续另外60分钟。 直接单层上皮损害，由于拉伸 2间接机制。一些研究表明，过度的机械牵拉肺上皮细胞可能会导致涉及细胞损伤11,13,26,27直接伤害。这种类型的伤害是难以机械腹胀期间没有捕捉实时成像。这样，利用所述定制装置和相差显微镜上连续的图像的细胞进行机械拉伸，记录之前和用促炎细胞因子（50毫微克/毫升的TNF-α为6小时）（ 图5）处理之后。细胞领域相同的图像被抓获提高应变水平。图像显示当细胞10和15％的应变之间伸展的红色箭头所示形成间隙。要注意，这些图像被记录在接近实时，&lt;10秒的滞后的伸缩的端部之间是很重要的<html>小时，成像，同时所述细胞拉伸。在拉伸细胞的细胞成像以往的研究，图像前后拉伸比较细胞中，只有图像不拉伸时病情11,13,28获得。 在拉伸细胞AFM纳米压痕 该机械装置被放置在与使用一个适配器板的原子力显微镜（ 见图6A）。获得（ 图6D-E）采用先前公布的方法13，弹性模量地图（电子地图）MLE12细胞前和拉伸后，10％的应变。虽然被在10秒内的拉伸而得到在10％应变（ 图6D）相衬图像，在图6D-E中所示的电子地图消耗大约22分钟，记录在一个40微米×40微米的300个人力和变形的曲线用在z方向上的末端的2.5微米/秒的速度的区域。OM /文件/ ftp_upload / 52737 / 52737fig1highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图1" src="/files/ftp_upload/52737/52737fig1.jpg" /> 图1.定制设计的装置用于细胞培养和伸展。 （A） 的设备的机械设计的计算机生成的图，（B）的已完成的装置中，（C），连接到所述马达和线性驱动器，其将电动机的旋转转换为1三维移动装置的照片的照片其中控制夹具双轴拉伸（D）硅橡胶膜。该基板包含每个夹子标签上两个孔放置在每个夹子的职位。柔性膜，它是整个结构的四分之一 ，由于使用的对称边界条件（E）的表示尺寸的有限元模型。 （ <s<html>仲&gt; F）最大主应变显示在全膜描绘表示各向同性应变场圈的形成。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52737/52737fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52737/52737fig2.jpg" /> 图2.（A）的支承的柔性膜与施加到测量外加应变标记的装置被示出。特写图像的标记，前（B）和后（C）的拉伸，分别标有箭头，以清楚地指示在膜上的标记的位移。 （D）膜作为应变电机的功能在逼近和退回计数。膜株均逼近和退回与回缩生产略高株相似。<html> HREF ="htt​​ps://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52737/52737fig2highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52737/52737fig3.jpg" /> 图3.膜应变传输到细胞中。（A - B）上使用10X物镜，柔性膜的人类支气管上皮细胞（16HBE）的前（A） 的相位对比图像和（B）20％的应变后。 （C - D）的16HBE细胞的荧光标记的吲哚（DAPI）细胞核用20X物镜共焦显微镜下，前（C）和（D）的 20％的应变后成像。 （E）的膜所经历的应变的细胞是线性和均匀。T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52737/52737fig4.jpg" /> 图4.使用使用共聚焦显微镜下一个20X物镜的累积线粒体超氧化物传感器观察单个拉伸的ROS产生16HBE细胞的影响（A - C）快照的时间过程（0，60分钟，并〜超生产的弹力细胞的同一领域中65分钟）。 （ 四）16HBE细胞的相对荧光强度随着时间的推移表明荧光强度增加了50％，从基准生产超的第一个小时内（A到B）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52737/52737fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图肺泡上皮（MLE12）单层应对日益舒展5.相衬图像（A - D）。采用数码显微镜20倍的目标的所有记录。箭头指明细胞-细胞分离发生的位置。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52737/52737fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52737/52737fig6.jpg" /> 图6（a）机械在AFM的纳米压痕细胞伸展的显示设备。扫描器必须为放置目标扩展器被除去。三腿增量被附加到所有三条腿的原子力显微镜头，以允许装置安置。一接合板是利用安装在担架上的原子力显微镜扫描仪（黑色板）。悬臂梁扫描中的前伸展相衬图像的MLE12电池单体（B）中所示的箭头的方向。红色方块的前（B）和后（C）的拉伸图像描述被在纳米压痕（40X物镜）捕获的区域。弹性模量的地图，之前（D）后（E）伸展，与此前公布的分析技术得到13。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52737/52737fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

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 ...

live-cell-imaging-during-mechanical-stretch

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.3K 次观看.  University of Tennessee Health Science Center. 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. 

视频: Live Cell Imaging during Mechanical Stretch

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

采用循环单轴应变的干细胞的机械性刺激

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

科研

生物学

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

在胎儿肺细胞的机械传导的实验系统研究

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

生物工程

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

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 ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

神经科学

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

人隐静脉内皮细胞分离和暴露于受控水平的剪切应力和拉伸

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...