作者要感谢查尔斯·基斯博士，基督教伦肯博士，基督教Dehnert（应用生物物理学Inc。）和乌尔夫拉德勒尔博士（ibidi有限公司）为他们出谋划策，帮助和这份手稿的准备过程中的富有成果的讨论。再者，我们要感谢扬·贝祖·他出色的技术支持。

1。测量系统的研制<ol><li>开始实验，以防止结露之前在孵化器阵列架Prewarm至37℃。 </li><li>装满蒸馏水的保育箱的水棺材以避免井的干燥。 </li><li>执行对细胞和数组的所有操作在层流柜的无菌条件下进行。 </li></ol> 2。阵列细胞和接种的制备 注：ECIS阵列需要清洁和稳定实验之前，以防止电极漂移，提高富裕重复性好和信号噪声比。因此，有两种选择：用L-半胱氨酸和B）的ECIS的稳定功能电极A）预处理。 选项A：L-半胱氨酸被推荐为最稳定的预处理，但可能在一些特殊情况下与蛋白质相互作用涂料-GS上的电极。呈现的卷用于标准的8孔阵列。 <ol><li>添加200μl/孔的10mM的L-半胱氨酸。半胱氨酸清理和修改电极表面提供了高，重复性好电极电容。 </li><li>孵育15分钟，在室温（RT）。 </li><li>用超纯水（类型1）洗两次500微升/孔。不使用磷酸盐缓冲液，其可与某些蛋白质的吸附造成干扰。此外，还要避免含血清的涂层解决方案之前，因为在金表面会吸附在接触带来的第一个大分子。 </li><li>大衣与200μl/孔暖1％的明胶。 </li><li>孵育30分钟，在37°C。 </li><li>除去明胶，但不要让表面干燥。这可能会损坏电极。 </li><li>用超纯水（类型1）洗一次。 </li><li>加入400μl/孔的完整的细胞培养基（含血清，抗生素和所有的生长因子）。 </li><li>幻灯数组转换成数组的持有人d确保该阵列定位，使得金九方垫是由弹簧加载的弹簧针和定位调整螺丝用手拧紧正确接触。小心与透明阵列，金层很容易被损坏。 </li><li>打开测量软件，并按下设置其次是检查 “收集数据”一节中进行各单井的快速阻抗测量。所得到的值是测量的基线，并会自动被存储在“评论”。 </li><li>该井检查会自动判断使用ECIS阵列的类型。验证“好配置”一节中所选择的数组。 </li><li>该阵列图亮起绿色为正确连接井和红色为空或不正确的连接孔。不过，始终确保了阵列，在支架放置正确。 注意：如果清洁和涂层是成功的8W1E ECIS阵列有交流apacitance约5 nF和50 nF的，这导致在约2000和200Ω的基线阻力分别8W10E阵列。 </li><li>从支架上取下阵列。 </li><li>种子细胞如单细胞悬浮液中加入400μl/孔的完整的细胞培养基。 </li></ol> 方案B：在ECIS的稳定功能可以用作与L-半胱氨酸处理的替代或组合地，如果问题的，电极漂移或在油井到井电阻和电容的大的差异发生。 <ol><li>执行半胱氨酸治疗（可选）和预涂层电极与蛋白质（可选）。 </li><li>添加完全培养基200μl/孔，每孔放置在阵列夹持器中的阵列。 </li><li>打开测量软件，并按下设置其次是检查 ，以确定阵列已正确连接，并测量初始Z，R和C。 </li><li>按稳定电极<sTRONG&gt;稳定的“收集数据”部分。企业家信心指数将适用高电流（ 约 3毫安），高频率（64千赫）脉冲来清洁电极。 </li><li>再次检查电极。电容应增加和电阻值应该在可比较的范围。 </li><li>如果电容和电阻值仍然不理想，重复的稳定。 </li><li>从支架上取下阵列和接种细胞。 </li></ol> 3。设置测量软件和数据采集<ol><li>放置在阵列的阵列夹持器中，如前所述。 </li><li>打开测量软件，并按下“收集数据”部分中的设置按钮，ECIS的软件连接到ECIS的仪器和运行另一家检查 。 </li><li>验证“好配置”一节中所选择的数组。 </li><li>选择井在“好CONFIGURAT来衡量离子“一节。 </li><li>从“收集数据”选项中选择测量模式： <ol><li>对于一个时间序列在一个固定的频率，选择单一频率。 /时间（SFT），然后从下拉菜单中选择测量频率。 </li><li>对于电极覆盖和Rb和阿尔法的建模测量，选择多个频率。 /时间（MFT）。 ECIS的设备将在所有可用的频率自动测量。 </li><li>对于微动（见数据分析）选择快速采集时间（RTC）和调整测量频率和采样频率。这里的标准时间分辨率为每秒（1赫兹）一个样品，但取样频率也可以增加，以跟踪阻抗高达25赫兹（用于Z-θ）的快速变化。重要提示：在RTC模式只是单一的测量。 注意：如果微动需要在多口井进行后续计量，转到帮助&gt;显示专家工具条/菜单项&gt; ACQuire&gt;多井RTC。在收集数据部分指定的时间限制（小时）; ECIS的现在测量的数字顺序选定井。开始测量后，软件会要求指定的周期数。输入频率的测量需要重复的值。 </li></ol></li><li>对于SFT和生产厂商，选择测量或之间的时间间隔（秒），如果数据需要尽可能快地取消选中此选项获得。 注：要采集数据ECIS的设备使用一个多路复用器从一个0.25秒的最低SFT采集速率和7.5秒，MFT转好到另一个。意的时间间隔是依赖于所选的井和频率的数目。对缓慢增殖细胞的测量或电阻与时间的关系的一个简单的记录，可以使用600秒以上的间隔。 </li><li>按开始键，将文件命名并选择存储数据的位置。 注：文件大小不应该是一个公关oblem，因为ECIS的数据被保存在一种名为*。ABP纯文本格式，它决不会超过几百MB甚至与所有的测量频率和选定的井的数量。 </li><li>停止按完成运行。 </li></ol> 4。媒介变化与细胞操作<ol><li>按下暂停 ，这将停止数据采集，而是继续运行实验时钟。 </li><li>从支架上取下阵列和在层流工作台（ 即改变介质）操纵它。 </li><li>将在后面的支架阵列，要么单击检查连接或恢复实验继续进行数据采集。测量软件将放置一个时间标记的数据集。 </li></ol> 5。急性刺激​​过程中的数据采集注：第二个选项是数据采集遵循瞬时变化过程中操纵细胞。这是唯一可能的;当样品不必须是无菌的实验的进一步过程。 <ol><li>刺激直接添加到井，并确保混合小心用200微升移液器。 </li><li>马克时间按标记手动点并添加评论。 </li></ol> 6。电气伤人注意：寻找为使用的细胞类型正确的设置是关键电气伤人。如果伤人太短可能会导致去除细胞的不足，而如果伤人过长和/或粗糙，否则会损坏电极（尤其是在透明数组）。因此，几个短脉冲伤人建议。对于脐静脉内皮细胞培养2 10-20秒长伤人为5 V，60 kHz的脉冲，每个已发现最佳的使用ECIS 1600R。一般来说，建议使用高频率&gt; 20千赫保持整个细胞均匀的高场，并避免在电极上的任何损坏。 <ol><li>执行伤人DURing一个积极的ECIS的测量。 </li><li>使伤口/电穿孔设置 ，然后单击伤口 ，并填写时间（秒），伤口电压（V为1,600 R）或电流（μA为Z和Z-θ）和频率（Hz）。所显示的默认值基于阵列和ECIS的设备的类型。 </li><li>选择井“好配置”一节中的伤口。只有检查井将被打伤。 </li><li>启用延迟直到一小时 ，并填写延迟时间，以激活延迟伤口的功能。此功能可以通过按停止或手动应用伤口停止在任何时候。只有一个延迟指令可以同时被激活。 </li><li>按下启动 ，然后单击确定 ，在弹出的窗口中启动伤人。 </li><li>确保电阻信号下降到伤人后偏移阻抗，否则重复伤人。 </li></ol> 7。数据分析</P&gt; <ol><li>数据表示和出口。 <ol><li> &gt;打开-通过文件设置完成测量或加载数据。 </li><li>选择井从“好配置”窗口中显示。集团单井呈现的平均值。 </li><li>选择参数可从上部工具栏（Z，R或C）显示。 </li><li>从上面的工具栏（T =时，F =频率，3D =瀑布印迹）选择图表类型。 </li><li>定义图形偏移和范围与图形窗口下方的滑块或填写相邻控件中的精确值。 </li><li>从基本的“时间序列选项”中的“分析”部分执行标准偏差在选择，运行数据的平均值或正常化。 </li><li>启动专家工具栏像Nyuist情节和傅里叶变换更高级的操作帮助菜单中。 </li><li>使用“显示选项”中的“分析”一节中定义RANGE x轴和y轴和输出图形。 </li><li>导出所选数据通过文件 Excel文件- &gt;导出数据- &gt;到Excel（部分），并根据需要选择使用的第三方软件的深入分析，统计和代表性。 </li></ol></li><li> Rb和阿尔法的造型。 注：对于建模，建议使用无细胞，培养基填充和参考。 <ol><li>完成MFT测量或负荷生产厂商的数据集。 </li><li>如果无细胞很好的参考可以按任一查找 ，自动选择参考值或设置为手动从“频率扫描建模”中的“分析”部分中选择该单元格的空闲时间点。 </li><li>如果没有无细胞的参考，从另一个数据集导入的参考值。打开引用数据集（请确保相同的数组和培养基使用），并使用Find或设置为选择的参考价值。然后定位到测量数据，按获取 。 </li><li>如果没有无细胞引用数据集可使用出厂默认值。 </li><li>按模型开始计算。模型可能需要几分钟依赖于数据集的大小。 </li></ol></li><li>计算微动的。 注：此计算是不测量软件的一部分，但可以与任何信号处理软件来执行。 <ol><li>完成RTC测量或加载RTC数据集。 </li><li>归一化数据的整个期间的平均电阻设置。 </li><li>打破数据套入段与256个数据点的长度，并用汉宁窗清洁各分部。 </li><li>运行一个256点傅立叶变换，得到的平均频谱，以单面129点的功率谱，以减少随机噪声和增强的生物学信号。 </li><li>暗算幅度在双对数曲线图，并应用频率最小二乘法线性拟合。 </li><li>的线性拟合的斜率是在信号中总的噪声，从而微动的细胞的度量。 </li></ol></li></ol>

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应用生物物理学公司涵盖了开放接入费用，而结构和文章的内容仍是作者承担全部责任。

ECIS是一个极好的工具，用于细胞的属性和行为的审查以及对已知和未知物质的影响进行量化。从而将细胞的标准培养条件下保持，阻抗可以连续地以高时间分辨率的监视和相关的光学信号。该方法用于细胞操作的最佳时间点，可以选择的形态和功能的细胞状态的基础上。不幸的是，这种高测量分辨率自带的价格，在温度，pH值或单元格的机械刺激（中等城市）的微小变化会立即影响到阻抗信号。 在细胞上的小测量电流的应用使得ECIS测量无创，无损和无标记的，但作为结果只有被动生物电特性可以被测量（无动作电位）。一个重要特点是，一些参数可以是DER<html>从一个单一的测量IVED，从几个经典实验，如渗透率或伤口愈合实验相结合的信息。这里特别感兴趣的方面是，从数学建模的数据可以用来探索变化的电阻和电容，并将它们指的是不同的细胞结构（ 例如细胞接触或细胞膜）。重要的是要注意的是，阻抗谱总是在感测电极，其不容许在单细胞研究，也对数学模型是唯一有效的在汇合的细胞层提供来自所有小区的平均信号。因此，内皮细胞应在合流状态保持之前用于建模，以确保成熟的细胞粘连和静止期细胞至少一天。同样，电气伤口应只适用使用多伤人短脉冲高频率汇合的细胞层，以达到最佳的伤人效率和防止电极损坏。 jove_content“&gt;为了从ECIS测量得到的信息的最大数量，如在每一个试验中，几个参数，如阵列基板，涂料，接种密度为个别细胞类型的组合，需要进行测试和优化实验之前。 ECIS的一个主要限制是测量不提供分子水平上的直接信息。因此，ECIS的测量通常是最翔实的实验系列，以帮助联想一个科学问题与细胞结构或特性，并提供一个可检验的假说的产生显著输入的开头。因此，ECIS的模块化设计提供了广泛的应用与可能性量身定做的数组。最新的阵列预示未来专注于高吞吐量的阻抗筛检细胞增殖和电气伤人及特殊流动阵列在体内模拟的前进</eM&gt;剪切应力与不同的流量分布。 此外文献 请同时参阅应用生物物理学的网页（www.biophysics.com）的应用笔记，网络研讨会和出版物涵盖整个ECIS谱的详细清单。

ΩΩΩHere我们提出电动细胞基质阻抗传感，被称为ECIS，测量和分析贴壁细胞在培养1的阻抗谱的具体方法。此协议的目的是提供这种特定类型的基于阻抗细胞分析的使用一个普遍适用的指导，对于一些从不断越来越多的应用程序的关键功能提供了协议。重点将是对细胞增殖，屏障功能，细胞连接和细胞运动的研究。 由于ECIS及其相关模型转换阻抗谱数据的生物学相关的参数是在其目前的形式介绍给科学界通过Giaever和基斯在1991年2，它经常被简称为TEER的测量（反系统上皮电阻），这是不准确的。差异似乎缘在第一，但是对数据的解释很重要。对于经典TEER测量，细胞生长在可渗透的过滤器来表征细胞旁转运的机制，这是由上皮细胞紧密连接或内皮粘着连接3为主。常见的是，位于上方和下方的过滤器的两个电极用于施加一个直流（DC）流过电池层和另外两个电极来测量所产生的电压降4。的电阻是使用欧姆定律，它允许细胞屏障的质量的数值描述计算。 ECIS遵循这一基本原则，并扩展它。在ECIS系统，细胞生长在嵌入在特殊细胞培养皿的底部相对的，圆形的金电极。每培养电极的数量以及是可变的，依赖于应用程序和电极有250μm的标准直径，在某些情况下，较大的反电极用来完成电路。 ECIS使用1μA与一个给定的频率，而不是直流电流的恒定交变电流（AC）。的阻抗是由电极之间的电压的相应变化（以mV为单位）计算。 ECIS提供测量在一个频率范围，研究频率依赖性细胞的性质，这有一些优点TEER和本文中详细地进行说明的阻抗的可能性。首先，测量复阻抗允许分离的整体阻抗为细胞屏障电阻和电池电容。此外，通过采取数据在多个频率和施加的数学模型，可以交界阻抗（的细胞 - 细胞接触紧密度）和阻抗引起的细胞 - 基底相互作用（距离基底细胞膜到底层矩阵的），以及区分细胞膜电容的贡献。第二，细胞增殖和活力进行评估，由于细胞s为与电极直接接触。第三，基体和电极是足够薄，以允许对明场和相衬显微镜。 阻抗测量基准：复阻抗依据生物对象（ 例如细胞）的电阻抗的测量是欧姆定律，一个基本的电子技术原理，它描述了电阻（R）的关系，电流（I）和电压（U）中的电路在给定的时间（t）。 适用于直流电路：R（T）= U（T）/ I（T） 在AC系统中工作时，电流和电压，不仅在它们的振幅，而且在它们的相位（φ）。现在，阻力不再足以说明这些关系。取而代之的是，复杂的阻抗（Z）或阻抗在大多数情况下的幅度（| Z |）时，含有先前所描述的欧姆电阻加电抗（X），从而重新sults从交流电流经电容器和电感器的驱动电压和电流5之间的相移。 适用于交流电路：| Z（F）| =√（R 2 + X（F）2） φ=反正切（X / R） 当对完整细胞中，由于它们的膜的特性阻抗的测量，细胞作为电阻器和电容器的并联连接。这里，电阻表示反对电流流动，而电容（C）描述的电载流子在绝缘双层细胞膜，导致极化的细胞的分离。从而X由细胞膜的电容性能的支配。 X（F）≈（2 * PI * F * C 电池 ）-1 因为X是依赖于频率，测量频率的变化使之细胞的不同功能和结构性质的研究。 ECIS的设备测量R和X，使计算的| Z |，C和φ。 量化整个细胞层与阻抗谱：电气等效电路。 如前面所解释的，当一个细胞被带入一个电场，它示出了无源电子元件的性能。如果现在，而不是一个单一的细胞，生长在电极顶部和补充细胞培养基的整个细胞层进行了研究，需要电阻器和电容器的简单模型被扩展到整个电网。在此所谓的等效电路中，培养基（R 医务 ）以及电容（C ELECTR）和电阻（R ELECTR）特征的电极/电解质相互作用的电阻需要考虑3,6。 这样的等效电路，用于贴壁生长的细胞层的简化，一般的例子可以在图1中找到。这样的一个数学的优势pproach来描述一个生物系统是那些电路可以细化自由采食和调整到特定的实验问题， 例如 ，通过考虑阻抗引起的胞内细胞器或区分的细胞-细胞的影响（R JUNC）和细胞-基底粘连（R 分 ）对整体阻抗7,8。尽管如此，目的为造型应该总是使用最小的数字描述了测量阻抗频谱的所有功能，让有意义的相关性元素。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51300/51300fig1highres.jpg" width="500" /> 图1。示意图ECIS的系统和有代表性的等效电路附着生长的细胞层的ECIS的文化以及A）的横截面。将细胞生长于传感和反电极和一个顶重新盖上培养基。该电极被连接到一个锁定放大器和一个AC信号经由1兆欧电阻器施加以创建一个恒流源。刺激可以直接加入到培养基中在时刻B）ECIS措施的阻抗所有个体贡献的总和的任意一点。培养基（R 医务 ）以及阻抗所引起的电极/电解质界面，这是为了简单起见，作为一个电阻（R ELECTR）和一个电容（C ELECTR），并且还电性能的并联组合的电阻细胞膜，通过电阻的并联连接（R 单元 ）和电容（C MEM）中描述的，所有需要被考虑。 Ř 细胞是可变的，因为它是依赖于对当前的细胞渗透性。的等效电路可以扩展和细化自由采食 。作为一个例子交界（R JUNC）为以及内皮下（R 分 ）电阻被添加到电路。 <a href="https://www.jove.com/files/ftp_upload/51300/51300fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 阻抗参数及其生物学意义从阻抗测量得出的两个最直接的参数是电阻和电池的容量。电阻代表质量和小区屏障的功能，因此，考虑到对对 - 和反式蜂窝电流流动的电阻。电容可提供电极覆盖的整体措施。 ECIS的的显着特点是，与等效电路的帮助和建模的全局参数提供更多的细胞特性，包括细胞 - 细胞和细胞 - 基底粘连，这将在本文后面讨论的见解。 实验conside：在开始之前口粮 测量设置 -该设置由几个独立的部分组成：ECIS的设备与测量仪表，PC机进行数据采集;为8阵列持有人-或96孔系统，ECIS阵列和选择的细胞培养。阵列夹持器必须放置在温箱中，并连接到所述培养箱外ECIS的设备。电脑需要配备ECIS的软件（1.2.123.0 2013年2月14日），并连接到ECIS的设备。 阵列的选择 -有一个不断增长的各种ECIS阵列，专为多种应用。标准阵列是8W1E和8W10E阵列，这是由8个培养孔（用W表示）分别包括1个或10个测量电极（用E表示）。一个大的反电极就完成了电路，但它的阻抗是在实际测量6基本上可以忽略不计。标准的8孔阵列架可以承载两​​个rrays，导致16个培养孔的总数量。金电极是50nm厚，划定有绝缘膜和安装在任何光学透明的Lexan聚碳酸酯基片或印刷电路板（PCB）。在PCB阵列更强大和成本效益。透明的幻灯片允许光线和免疫荧光显微镜。什么是必须考虑的是，1E阵列增强所造成的细胞运动的阻力信号不稳定，而且是需要伤口愈合的研究。此外，单个电极允许电信号和光信号的相关性。在多电极阵列，该信号被平均超过几个电极，它由于提高了测量区域包括多个小区在测量中，由不平坦的接种和细胞生长的限制数据的偏差，并减少了信号的晃动，由于细胞运动。因此，多电极阵列来研究细胞的增殖和势垒形成是有用的。下一步，标准阵列有可用于剪切应力9应用特殊的阵列，研究趋化10，细胞迁移和增殖，以及96孔板高通量筛查。最后，将用于在阵列强烈地依赖于科学问题和细胞类型和应选择和仔细测试。 测量频率 - Rb和阿尔法的造型（见数据分析）需要多频率测量（MFT）。否则阻抗可以在一种细胞类型的特定频率（SFT）来测量随着时间的推移，与该数据可以被收集具有较高的时间分辨率的优点。最灵敏的测量频率为特定的细胞类型可以通过频率扫描中找到。当在一个数 - 对数图中的频率分别绘制阻抗的电阻对频率的特性，其中间无细胞和细胞覆盖的电极是最大的区别是，其中所述细胞块T的频率他目前最有效的。若血管内皮细胞（EC）的这个频率是在大约4千赫。 接种密度 -由于在每一个定期的基于细胞的实验的接种密度依赖于科学问题。当研究的粘附和铺展或屏障形成细胞，内皮细胞应与40,000-60,000个细胞/ cm 2的高密度被接种，以保证汇合，48小时后是稳定的屏障。如果该实验的重点是在增殖，内皮细胞应接种大约2000-10000个细胞/ cm 2的密度低。

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			<td height="21" style="height:21px;width:255px;">Name of Material/Equipment</td>
			<td style="width:320px;">Company</td>
			<td style="width:247px;">Distributor Europe</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:329px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:255px;">ECIS Z&#952;</td>
			<td style="width:320px;">Applied Biophysics Inc., Troy, NY, USA</td>
			<td style="width:247px;">ibidi GmbH, Munich, Germany</td>
			<td>71617</td>
			<td><a href="http://www.biophysics.com/products-ecisz0.php">http://www.biophysics.com/products-ecisz0.php</a></td>
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			<td height="21" style="height:21px;width:255px;">8W1E PCB Electrode Array</td>
			<td style="width:320px;">Applied Biophysics Inc., Troy, NY, USA</td>
			<td style="width:247px;">ibidi GmbH, Munich, Germany</td>
			<td>70004</td>
			<td><a href="http://www.biophysics.com/cultureware.php">http://www.biophysics.com/cultureware.php</a></td>
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			<td height="21" style="height:21px;width:255px;">8W1E Lexan Electrode Array</td>
			<td style="width:320px;">Applied Biophysics Inc., Troy, NY, USA</td>
			<td style="width:247px;">ibidi GmbH, Munich, Germany</td>
			<td>70001</td>
			<td>http://www.biophysics.com/cultureware.php</td>
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			<td height="21" style="height:21px;width:255px;">8W10E PCB Electrode Array</td>
			<td style="width:320px;">Applied Biophysics Inc., Troy, NY, USA</td>
			<td style="width:247px;">ibidi GmbH, Munich, Germany</td>
			<td>70010</td>
			<td>http://www.biophysics.com/cultureware.php</td>
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			<td height="21" style="height:21px;width:255px;">L-Cystein</td>
			<td style="width:320px;">Merck Millipore</td>
			<td style="width:247px;">-</td>
			<td>102838</td>
			<td><a href="http://www.merckmillipore.com/germany/chemicals/l-cystein/MDA_CHEM-102838/p_GLOb.s1L7V8AAAEWL.EfVhTl?WFSimpleSearch_NameOrID=L-cystein&amp;BackButtonText=search+results">http://www.merckmillipore.com/germany/chemicals/l-cystein/MDA_CHEM-102838/p_GLOb.s1L7V8AAAEWL.EfVhTl?WFSimpleSearch_NameOrID=L-cystein&#38;BackButtonText=search+results</a></td>
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			<td height="21" style="height:21px;width:255px;">Gelatin</td>
			<td style="width:320px;">Merck Millipore</td>
			<td style="width:247px;">-</td>
			<td>104070</td>
			<td><a href="http://www.merckmillipore.com/germany/chemicals/gelatine/MDA_CHEM-104070/p_S7Wb.s1LadYAAAEWaOEfVhTl?WFSimpleSearch_NameOrID=gelatine&amp;BackButtonText=search+results">http://www.merckmillipore.com/germany/chemicals/gelatine/MDA_CHEM-104070/p_S7Wb.s1LadYAAAEWaOEfVhTl?WFSimpleSearch_NameOrID=gelatine&#38;BackButtonText=search+results</a></td>
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			<td height="21" style="height:21px;width:255px;">Bovine Thrombin</td>
			<td style="width:320px;">Merck Millipore</td>
			<td style="width:247px;">-</td>
			<td>604980</td>
			<td><a href="http://www.merckmillipore.com/germany/life-science-research/thrombin-bovine-high-activity/EMD_BIO-604980/p__pmb.s1LNsYAAAEWS2IfVhTm?PortalCatalogID=merck4biosciences">http://www.merckmillipore.com/germany/life-science-research/thrombin-bovine-high-activity/EMD_BIO-604980/p__pmb.s1LNsYAAAEWS2IfVhTm?PortalCatalogID=merck4biosciences</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Albuman (Human Serum Albumin)</td>
			<td style="width:320px;">Sanquin</td>
			<td style="width:247px;">-</td>
			<td></td>
			<td><a href="http://www.sanquin.nl/en/products-services/plasma-products/">http://www.sanquin.nl/en/products-services/plasma-products/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Y-27632</td>
			<td style="width:320px;">Tocris Biosciences</td>
			<td style="width:247px;">-</td>
			<td>1254</td>
			<td><a href="http://www.tocris.com/dispprod.php?ItemId=1382#.UbiFNNish8E">http://www.tocris.com/dispprod.php?ItemId=1382#.UbiFNNish8E</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EGM-2 BulletKit&#160;</td>
			<td style="width:320px;">Lonza</td>
			<td style="width:247px;">-</td>
			<td>CC-3162</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/endothelial-cell-growth-media-kits.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/endothelial-cell-growth-media-kits.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pen/Strep</td>
			<td style="width:320px;">Lonza</td>
			<td style="width:247px;">-</td>
			<td>17-602E</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx">http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx</a></td>
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</table>

electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility

电动细胞基质阻抗传感（ECIS）是一种体外阻抗测量系统来量化贴壁细胞层中的细胞的行为。为此，细胞生长在上置，圆形的金电极的顶端特殊培养室。一个常数小的交变电流在被测量的电极和电势之间。细胞膜的绝缘性创建朝向电流流动导致在电极之间的增加的电势的电阻。以这种方式测量细胞阻抗允许细胞附着，生长，形态学，功能和活力的自动学习。虽然ECIS测量本身很简单，简单易学，基础理论是复杂的，选择正确的设置和正确的分析和解释数据是不言自​​明。然而，一个明确的协议，描述从实验的各个步骤设计，准备，实现和实验分析不可用。在本文的基本测量原理以及可能的应用，实验的考虑，优点和ECIS系统的局限性进行了讨论。提供了一种用于细胞附着的研究的导向，扩散和扩散;中汇合层，对于屏障功能，细胞运动，细胞 - 细胞和细胞 - 基底粘连质量细胞行为进行定量;和伤口愈合的量化和细胞对血管活性刺激物。代表性的结果是基于人微血管（MVEC）和人脐静脉内皮细胞（HUVEC）所讨论的，但同样适用于所有粘附生长的细胞。

设置实验是相当简单和测量本身是完全自动的，但是这是常有的情况下，正确的分析和对数据的解释是至关重要的。在代表性的数据部分的指南提供了一种通过阻抗谱与ECIS提供的最重要的参数的解释。这将有助于决定哪种科学问题的ECIS的测量可以是有用的，哪些参数应记录。 一个典型的实验读出可以普遍处于增长期细胞接种和高原相当细胞达到汇合后进行划分。每个阶段会产生不同的细胞属性的信息。代表性的测量， 如图2所示，并划分为四个子阶段来分析A）细胞粘附，铺展和增殖，B）形成了成熟欧共体屏障; C）细胞迁移电动的后一代人的伤口，以及D）与血管活性剂的凝血酶刺激的细胞应答。在不同的子阶段直接购入从测量电极相衬和免疫荧光图像，以说明电极覆盖范围和小区状态，并记录阻抗信号之间的关系。 黏附，伸展和增殖每个细胞类型都具有其特有的粘附和生长曲线，可以通过改变接种密度，与细胞外基质蛋白或其它刺激物涂覆被操纵。一，研究这些过程的主要困难是附着力区分，传播和扩散。韦格纳等 11给出了使用电阻和电容的参数和图3中可清楚如何电阻和电容可以互为补充来区分的组合的详细描述。它是重要密合性的研究，并蔓延到理解的测定频率的影响，因为这里的电场使细胞悬浮液8，迫使电流只流周围的细胞的细胞膜的极化。当该细胞附着在开始通过以线性的方式与开口面积对电极（分数面积）的比例散布在电极和电容减小，以限制电流流过。因此，粘附性，铺展和增殖可记录电容在频率高于40 kHz的（ 图3B），其中在电容的减小是直接正比于电极覆盖率较高时进行量化最好。可替换地在其最敏感的测定频率的电阻是直接测量的分化和细胞的成熟成汇合屏障（ 图3A）。除了 ​​部分区域，魏格纳等人提出的另外两项参数量化粘附和电极覆盖率：吨1/2（半最大电容），这是时间，直到电极的50％覆盖有细胞（ 图3B中 ，插入），和电容曲线（S，不斜率示出）的细胞扩散和扩散的速率。 电阻作为屏障质量的测度电阻阻抗描述屏障质量和功能最好的，因为它忽略了电容元件从膜，电极和介质的部分。这里的术语屏障质量和不透性时，由于磁导率是根据定义仅依赖于细胞 - 细胞接触。然而抗性是由细胞和它们的细胞 - 细胞和细胞 - 基质相互作用，以阻止电流流动的能力来确定。因此不同类型的细胞（克隆和通道），有其特定的电阻值（ 图4A）。虽然电阻给出了一个CLEARER想法对细胞屏障，阻抗信号应始终考虑。 Rb和阿尔法建模 ECIS的软件藏着一个特定特征，来模拟两个参数，细胞-细胞和细胞-基质的粘连（ 图4B和4℃）之间区分的能力。铷（以cm 2×欧姆），是细胞-细胞接触电阻对电流流动，从而古典渗透性的逆量度。高的Rb意味着对电流流动的低渗透性。阿尔法（以厘米×0.5欧姆），是用于从细胞-电极结所引起的阻抗的贡献的指标。定量细胞-细胞和细胞-基质接触模型被引入1991年由Giaever和基斯并基于这样的假设细胞是圆形的，具有一绝缘薄膜的盘形物体，悬停在电极和填充有传导性电解质2 。该绝缘细胞膜的属性只留下3通路的电流：a）该细胞和电极的腹面之间，b）该细胞 - 细胞接触，和c）通过细胞间（只可能当一个高测量频率使用）。更详细的推导和解释可在罗等人 3,12的论文中找到。 微动已经表明，该电阻信号（ 图4A）中的小波动是由于细微，随机细胞运动中汇合的细胞层以及细胞移动和关闭在稀疏培养2电极。由Lo和彩描述ECIS时间过程数据这方面等人 13,14常常被忽略，并与1E阵列和在最敏感的频率进行测量，以考虑对-和亚细胞的电流路径时，最清楚地看出。该单元格的计算造成噪声（微动）是不是个部分E标准测量软件，虽然在最新版本的快速傅立叶变换（FFT）被集成。记录RTC数据与1 Hz，持续1小时在4 kHz的取样频率提供足够的分辨率为EC微动的计算。该计算提供一个单一的值，可直接用于进行统计分析。对使用频率分析的微动扩展的讨论可以在其他地方15被发现。可替换地，以FFT分析其他策略都可以使用，如增量的方差。方差是对微动中的小变化更敏感，但由于这种敏感性的个体实验的比较变得困难。功率谱的斜率的微动的更稳定的测量（ 图4D）。这里的曲线下的面积可以被看作是微动或噪声的细胞建立的量。因此，较陡的功率频谱的斜率的曲线下面积较小和微动越小。 电动伤口愈合实验为了研究细胞的迁移，通常的机械伤口用移液器吸头，刮片细胞或特定邮票划伤细胞从培养板，并按照他们再迁移16显微镜应用。这种方法有明显的缺点，即从头开始的大小而不同，并且通常太大而忽略了对细胞增殖的伤口愈合过程的影响。 ECIS的的伤人函数使用高电压，高频率的脉冲，以获得无细胞电极，随后如下相邻的细胞来代替死亡细胞（ 图5A）的再迁移。以上标准，劳动密集的划痕测定这种自动化方法的一个优点是，ECIS产生高度可重复的伤口用250微米的精确直径，即在几个小时内关闭，以研究纯迁移。此外，电脉冲做上课不能去除蛋白质涂层和分泌细胞外基质。在这些细胞重新迁移对来自各方面的电极的假设下，迁移率可以很容易地通过将缠绕半径所要求的伤口缝合17时计算。作为替代电伤人，最近，所谓的电栅栏方法被介绍（未示出）。在这里，高电流脉冲防止粘附和细胞迁移的电极接种后。细胞将生长测量电极的周围，并开始向内迁移只要电脉冲被关断。电栅栏在电动伤人的优点是，在电极表面没有被改变由细胞生长或细胞去除，这是测量不同涂层对细胞迁移的影响很重要。 细胞刺激除了细胞迁移的研究，阻抗谱非常适合测量药物和细胞毒素的影响。 图5B呈现出经典的刺激/抑制实验，其中凝血酶被用来通过收缩和间隙形成单元，其表现为电阻的瞬时压降以诱导超渗透性的汇合EC屏障。通过预培养用Rho激酶抑制剂Y-27632的大部分凝血酶的反应是通过降低基底细胞张力18和阻塞应力纤维形成19减弱。然后该Rho激酶阻断剂被用来在不同时间点凝血酶添加后，导致EC屏障立即全面恢复。这里连续的阻抗测量系统来研究急性细胞反应的优点是显而易见的。在常规的端点检测（ 如免疫荧光染色或高分子通道），所使用的拦截这个急性反应将是检测和量化非常困难的，如果不是不可能的。重要的是要注意的是，与我mpedance测量对离子的通透性描述，而不是对大分子。详情请参阅阿曼等 20，其中提供ECIS的测量和大分子通过实验之间一个很好的对比。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51300/51300fig2highres.jpg" src="/files/ftp_upload/51300/51300fig2.jpg" /> 图2。代表性的测量。MVEC生长在明胶包被8W1E阵列5000个细胞/ cm 2和10天进行的MFT记录的低接种密度。测量说明了不同的读奏的ECIS的实验：A）黏附，伸展和增殖，B）形成和汇合细胞屏障成熟;后三）伤口愈合的伤口电器的应用，D）刺激的血管活性剂凝血酶。两个箭头表示媒体<html>点心，这是在4天的时间间隔进行，并给予有关的测量对机械刺激和变化，温度和pH敏感性的想法。在顶部面板上的图像对应的测量，并直接从测量电极收购。在左侧的相位对比图像显示了内皮细胞接种后24小时开始，以覆盖电极。在该中心，并在融合内皮细胞层的右侧现在的F-肌动蛋白染色免疫荧光图像之前和刺激凝血酶（1单位/毫升）后。由凝血酶形成所谓的应力纤维和内皮间间隙（箭头和图像插入），在识别ECIS的信号通过电阻下降到基线短暂开放会导致内皮细胞收缩。这里测量的灵敏度变得明显和如何变化，细胞层相关联的阻抗信号。s/ftp_upload/51300/51300fig2highres.jpg“目标=”_blank“&gt;请点击这里查看该图的放大版本。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51300/51300fig3highres.jpg" src="/files/ftp_upload/51300/51300fig3.jpg" /> 图3。细胞附着和生长。MVEC来自同一供体进行接种在明胶包被8W10E阵列和记录细胞生长在多个频率进行60小时3不同密度A）以4kHz的频率最佳电阻值的三个条件来测量屏障的形成。所有这三个播种密度成立ca的融合障碍。 1,200Ω在测量结束;增殖率不过（曲线的斜率）明显不同B）电容的测量在64千赫提供的粘附和铺展的见解。附着力是在免除expat原始高原期的电容曲线（2-8小时之间）。扩频，这是在向电极覆盖的线性关系，由曲线的斜率表示与取决于接种密度10-30小时后完成。在时间点t 1/2（半数最大覆盖范围），可用于统计分析。 <a href="https://www.jove.com/files/ftp_upload/51300/51300fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51300/51300fig4highres.jpg" src="/files/ftp_upload/51300/51300fig4.jpg" /> 图4。集流屏障的表征。两个MVEC献血者不同通道号（通道3和6）接种于明胶包被的8W1E阵列和执行了MFT的测量30小时来表征EC屏障。A）电阻<html>测量表明年轻供体1具有ca的较高的阻力。 11000Ω相比， 约 7,500Ω老施主2 年）的ECIS的建模功能已被用来寻找原因为较低的电阻。建模揭示了类似的细胞-细胞相互作用Rb和表示弱化的细胞-基质相互作用阿尔法作为一个可能的原因。D）RTC进行量化微动通过傅立叶变换，其中在曲线下的面积成比例的微动的汇合细胞层内。光谱的分析显示旧的捐助2少微动，从而支持一下已经可以从电阻信号（小于方差）承担。 <a href="https://www.jove.com/files/ftp_upload/51300/51300fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 5“FO：内容宽度=”5英寸“FO：SRC =”/ files/ftp_upload/51300/51300fig5highres.jpg“SRC =”/ files/ftp_upload/51300/51300fig5.jpg“/&gt; 图5。细胞迁移电伤人及凝血酶刺激的影响后，阻抗录音在4 kHz的执行与最大时间分辨率。 一）MVEC捐助1被用于通道3和捐助2在明胶包被8W1E阵列通道6和生长至汇合。然后电伤是由应用程序的两个5伏的脉冲，在60千赫与每个20秒的持续时间建立。年轻的供体1表现出更好的伤口愈合的基础上以更快的伤口闭合（迁移，该曲线的斜率）的能力，打伤后较高的电阻相比于较旧的供体2 B）血管内皮细胞生长在明胶包被的8W10E阵列。融合后的细胞已经通过用基础培养基中加1％人血清白蛋白（HSA）的更换完全培养基被血清饥饿1.5小时。我们普通中等与人血清白蛋白的年龄是关键的一步，因为血清成分阻断凝血酶的反应。只要一个稳定电阻高原检测，凝血酶直接加入到各孔中，并与200μl的移液管小心地混合。 30分钟后，其特征在于，在电阻瞬时下降到基线和一个CA后的最大凝血酶效应是可见的。恢复后降低30％-50％的电阻。将细胞预孵育或者与Rho激酶阻断剂Y-27632 30分钟或阻滞剂加20，30，或40分钟凝血酶添加后，立即减少凝血酶的作用。 <a href="https://www.jove.com/files/ftp_upload/51300/51300fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility at JoVE.com

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

此协议的评论电器细胞 - 基底阻抗传感方法来记录和分析的贴壁细胞的阻抗谱细胞附着，增殖，运动，和细胞反应的药理学和毒性刺激的量化。检测血管内皮细胞通透性和细胞 - 细胞和细胞 - 基质接触的评估被强调。

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

electric-cell-substrate-impedance-sensing-for-quantification

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56.6K 次观看.  Institute for Cardiovascular Research, VU University Medical Center. 此协议的评论电器细胞 - 基底阻抗传感方法来记录和分析的贴壁细胞的阻抗谱细胞附着，增殖，运动，和细胞反应的药理学和毒性刺激的量化。检测血管内皮细胞通透性和细胞 - 细胞和细胞 - 基质接触的评估被强调。 

视频: Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

科研

生物工程

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

细胞电微环境的有限元素建模

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

通过心血管磁共振成像定量小鼠心脏左心室功能、心肌劳损和血流动力学力

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

三尖瓣瓣叶的层显微切割，用于双轴力学表征和微观结构定量

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a chemotherapeutic might be functionalized with an antibody to enhance binding to cancer cells (&#34;targeting&#34;) or functionalized with polyethylene glycol to help evade immune cell recognition (&#34;stealth&#34;). Even at a cellular level, optimizing the binding and uptake of a drug carrier is a complex biological design problem. Thus, it is valuable to separate how strongly a new carrier interacts with a cell from the functional efficacy of a carrier&#39;s cargo once delivered to that cell.
To continue the chemotherapeutic example, &#34;how well it binds to a cancer cell&#34; is a separate problem from &#34;how well it kills a cancer cell&#34;. Quantitative in vitro assays for the latter are well established and usually rely on measuring viability. However, most published research on cell-carrier interactions is qualitative or semiquantitative. Generally, these measurements rely on fluorescent labeling of the carrier and, consequently, report interactions with cells in relative or arbitrary units. However, this work can be standardized and be made absolutely quantitative with a small number of characterization experiments. Such absolute quantification is valuable, as it facilitates rational, inter- and intra-class comparisons of various drug delivery systems-nanoparticles, microparticles, viruses, antibody-drug conjugates, engineered therapeutic cells, or extracellular vesicles.
Furthermore, quantification is a prerequisite for subsequent meta-analyses or in silico modeling approaches. In this article, video guides, as well as a decision tree for how to achieve in vitro quantification for carrier drug delivery systems, are presented, which take into account differences in carrier size and labeling modality. Additionally, further considerations for the quantitative assessment of advanced drug delivery systems are discussed. This is intended to serve as a valuable resource to improve rational evaluation and design for the next generation of medicine.

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type.

体 外药物递送系统和细胞之间相互作用的实验定量：临床前纳米医学评估指南

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a ...

转基因大豆毛状根的高效生产

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

作者聚焦：用于基因功能分析的大豆毛状根转化  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

大豆毛根转化基因功能分析

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...