使用具有可编程输出频率的自制伏特安度计执行 TEER 测量的简单方法
Summary
在这里，我们演示如何设置具有可编程输出频率的廉价伏特安度计，该仪表可与市售的筷子电极一起使用，用于跨皮/内皮电阻测量。
Abstract
自 20 世纪 80 年代以来，一直使用过皮/内皮电阻 （TEER） 来确定体外屏障模型系统的汇合性和渗透性。在大多数情况下，筷子电极用于确定包含蜂窝单层的细胞培养过滤器插入系统的上腔和下腔之间的电阻抗。过滤膜允许细胞通过建立紧密的结点粘附、极化和相互作用。该技术已被描述与各种不同的细胞系（例如，血脑屏障的细胞，血脑脊液屏障，或胃肠道和肺肠道）。TEER 测量设备可以从不同的实验室设备供应商处轻松获得。但是，如果适当的电压计是自行组装的，则有更具成本效益和可定制的解决方案。本出版物的总体目标是建立一个可靠的具有可编程输出频率的器件，该器件可与市售的筷子电极一起使用，用于 TEER 测量。
Introduction
上皮细胞和内皮细胞作为细胞边界发挥作用，分离身体的表皮和巴索侧。如果它们通过紧密的交汇点连接，则通过副细胞空间的被动物质扩散受到限制1，从而形成选择性渗透屏障。几个人工屏障系统已经开发了2使用微血管内皮细胞（HBMEC，血脑屏障3，4，5，6，7），乔体丛上皮细胞（HIBCPP/PCPEC，血脑脊液屏障8，9，10，11，12，13，14），结肠直肠腺癌细胞（Caco-2，胃肠道模型15），或气道/肺泡细胞系（肺模型16，17）。这些系统通常由在可渗透膜上的单层生长的细胞组成（即过滤器插入系统），以便进入锥形和双侧侧。模型系统的完整性与体内条件相匹配非常重要。因此，通过测量示踪化合物在细胞层中的准细胞扩散来分析阻物功能。这些物质包括放射性标记蔗糖，染料标记的白蛋白，FITC标记的inulin，或染料标记的德克斯特兰2。然而，化学染料会使细胞无法用于进一步的实验。为了非侵入性地监测屏障系统，可以使用2、18、19测量跨皮层/跨体电阻（TEER）。由于双极电极系统受电极-电解质接口处电极极化阻抗的影响，通常使用四极测量来克服这一限制20。底铺设技术是一个四端传感（4T），最早由威廉汤姆森（主开尔文）21在1861年描述。简而言之，电流由一对载流电极注入，而第二对电压感应电极用于测量电压降20。如今，所谓的筷子电极由一对双电极组成，每个电极都含有用于测量电压的银/氯化银颗粒和用于通过电流2的银电极。电阻抗测量在锥形和双侧隔间之间，细胞层介于两者之间（图1）。在外部电极上应用频率为 12.5 Hz 的方波信号，并测量由此产生的交流电 （AC）。此外，通过第二个（内部）电极对测量整个细胞层的电位下降。然后根据欧姆定律计算电阻抗。TEER 值通过乘以阻抗和细胞层表面积进行归化，通常表示为±+ cm2。
在有些系统中，电池和电极的排列方式更为复杂，但也基于 4T 测量原理，可与相同的测量设备一起使用。例如，插入过滤器的 EndOhm 系统包含一个腔室和盖，其同心电极与筷子电极的结构相同。电极的形状允许更均匀的电流密度流过膜，从而减少读数之间的变化。更复杂的（但也更准确）是一个Using室，其中一个细胞层分隔两个腔室充满了Ringer的溶液22。腔室本身可以用氧气、CO2或N2气体，并搅拌或补充实验物质。当发生穿过细胞层的电离传输时，可以通过组织附近的两个电压感应电极来测量电位差。此电压由放置在电池层旁边的两个带电电极消除。测量电流然后给出净电位传输和透皮电阻，这反映了屏障的完整性，可以确定22。TEER测量也可以应用于代表屏障组织模型23、24的片上体系统。这些系统模仿细胞的体内条件，通常由几种类型的细胞组成，它们相互堆叠在一层之上。
以下协议说明了如何设置具有可编程输出频率的具有经济高效且可靠的伏特表，与市售的测量系统相比，TEER 在 TEER 中不会产生统计显著性差异。
Protocol
1. 用于 TEER 测量的基本伏安度计组装 准备一个标准的USB充电器作为5V D.C.电源，USB延长线，微控制器，将用作可编程方波发生器，两个标准万用表，能够测量交流电和电压作为根平均平方（True-RMS），四根带香蕉插头的电缆、带 RJ14 母连接器的电话延长线，包括六根带内四根有线 （6P4C） 的引脚、两条短电缆、一个光泽端子、一个 120 kΩ 预电阻器、线端铁圈和焊接凸耳。所需的工具包括绝?…
Representative Results
为了比较自组装的电压计与市售的电压表的运行情况，记录了两个器件的电压示波器。 如图2 A所示，当使用1 kΩ测试电阻器进行负载工作时，参考仪器生成了振幅为80 mV、振荡时间为80 ms的方波信号，相当于12.5 Hz的频率。 相反，如果没有设置预电阻器，自?…
Discussion
在自制伏特计可用于日常操作之前，必须检查设备是否正常工作。在我们的例子中，40 ms（12.5 Hz）的半时振荡被编程，但有效振荡时间的结果是60 ms （16.7 Hz）。微控制器时间发射器的这种不准确对TEER测量没有可检测的影响。最好使用万用表之一的频率设置来确定实际频率。如果发现任何偏差，可以相应地调整源代码。此外，强烈建议检查测试电阻器或其他定义的设置是否提供正确且可重现的结?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢赫尔曼·利格斯迈尔和马文·本德在电工和信息学方面的专家建议。
Materials
| 120 kOhm resistor | General (generic) equipment | ||
| Banana plug cables | General (generic) equipment | ||
| Cables | General (generic) equipment | ||
| Chopstick electrode | Merck Millicell | MERSSTX01 | |
| Chopstick electrode (alternative) | WPI World Precision Instruments | STX2 | |
| Crimping tool | General tool | ||
| Digispark / ATtiny85 | AZ-Delivery Vertriebs GmbH | Digispark Rev.3 Kickstarter | |
| DMEM:F12 | Gibco (Thermo Fisher) | 31330038 | |
| Fetal calf serum (FCS)/Fetal Bovine Serum (FBS) | Life Technologies | 10270106 | |
| Filter inserts 3µm translucent | Greiner Bioone | 662631 | |
| HIBCPP | Hiroshi Ishikawa / Horst Schroten | ||
| Insulation stripper | General tool | ||
| Luster terminal | General (generic) equipment | ||
| Oscilloscope | HAMEG | Digital Storage Scope HM 208 | |
| Plotter | PHILIPS | PM 8143 X-Y recorder | |
| Software Arduino | https://www.arduino.cc | Arduino 1.8.9 | |
| Soldering iron | General tool | ||
| Soldering lugs | General (generic) equipment | ||
| Telephone cable with RJ14 (6P4C) connector | General (generic) equipment | ||
| Test resistor | Merck Millicell | MERSSTX04 | |
| True-RMS multimeters | VOLTCRAFT | VC185 | |
| USB charger | General (generic) equipment | ||
| USB extension cord | General (generic) equipment | ||
| Voltohmmeter for TEER measurement | WPI World Precision Instruments | EVOM | |
| Voltohmmeter for TEER measurement (alternative) | Merck Millicell | ERS | |
| Wire end ferrules | General (generic) equipment |
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Citazione di questo articolo
Theile, M., Wiora, L., Russ, D., Reuter, J., Ishikawa, H., Schwerk, C., Schroten, H., Mogk, S. A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency. J. Vis. Exp. (152), e60087, doi:10.3791/60087 (2019).