Name: fabrication and validation of an organ-on-chip system with integrated electrodes to directly quantify transendothelial electrical resistance
Uploaded: 2017-09-26T15:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance at JoVE.com

我们感激地感谢约翰引爆制造的模具和 Mathijs Bronkhorst 进行了卓有成效的讨论和协助数据表示。
这项研究的经费来自: SRO 生物医学微冷害 Segerink, 米拉大学生物医学工程和技术医学研究所, 特文特;特文特大学生物医学工程与技术医学研究所 Meer SRO 上;和 VESCEL, 紧急救济会向 A. van 伯格 (669768 号赠款) 提出。

1. organ-on-chip 设备的制作 <ol> <li> 使用标准的光刻技术制作了一个带有通道设计的模具的一个集成电路副本, 并获得了微流控芯片的硅氧烷部分。
	<ol> <li> 重约27克的硅橡胶基剂和2.7 克固化剂; 质量比率需要10:1。这是良好的3毫米厚的硅橡胶板上的模具与130毫米 (5 英寸) 直径。将这些组件彻底混合. </li>
		<li> 将混合物加在干燥中大约45分钟以除去气泡. </li>
		<li> 同时, 通过在模具周围粘贴透明胶片或将模具放置在合适的晶圆架上, 准备好液体硅烷混合物的模具. </li>
		<li> 将脱的硅橡胶混合物倒入模具上。如果任何气泡保持在该公司的混合物或在模具表面, 再次加气它约30分钟. </li>
		<li> 在60和 #176 的烤箱中固化硅烷混合物; C 为 4 h. 允许冷却. </li>
		<li> 在一个横流的引擎盖中, 从模具中拉出固化的硅橡胶; 可以立即重用该模具, 以制作更多的复制副本. </li>
		<li> 在集成电路中使用切割线将该复制副本切割成独立的顶部和底部芯片部件. </li>
		<li> 在顶部零件上打四孔, 使用1毫米直径的锐利穿刺活检, 形成入口和插座。从内到外打孔, 防止集成电路碎片在芯片中收集。用透明胶片覆盖芯片部件, 防止灰尘. </li>
		<li> 将聚碳酸酯膜从 Transwell 插入到大约3和 #215 的正方形中; 3 mm 2 . </li>
	</ol> </li> <li> 在两个硅橡胶部件之间组装多孔膜无泄漏, 以便组装与多孔膜接口的双层设备. 
	注意: 此协议是从 Griep et al 改编的。 19 和觉的 et al 。 26 <ol> <li> 使用0.7 克的硅橡胶基剂、0.07 克硫化剂和540和 #181; 甲苯的 L, 产生5:3 的重量比。彻底地涡旋砂浆. </li>
		<li> 自旋涂层200和 #181; 在六十年代的 1500 rpm 上, 砂浆上的玻璃片上, 以获得一层薄薄的均匀的砂浆. </li>
		<li> 将一层薄薄的砂浆从片上转移到底部, 然后用墨辊将其顶部移到芯片上。把底部的部分放在烤箱安全的盘子里. </li>
		<li> 用一组镊子将膜的边缘浸入自旋涂层砂浆中, 并将其小心地放在底部的中间部分. </li>
		<li> 小心地将顶部部分放在底部, 同时注意对齐. 
		注意: 不要将压力施加到芯片上, 也不要将顶部滑动到底部, 以防砂浆进入通道并堵塞膜. </li>
		<li> 用透明胶片盖住芯片入口, 防止灰尘进入芯片并在60和 #176 烘烤; C 表示3小时. </li>
	</ol> </li> <li> 将电极集成到侧面通道中. 
	注意: 此协议是从 Griep et al 改编的。 19 和 Douville et al 。 18 <ol> <li> 将铂金线切割成大约2厘米的碎片. 通过将它们浸入丙酮中30分钟, 以清水和乙醇冲洗, 并允许干燥。
		<li> 在一个横流的遮光罩, 把一个芯片上的塑料盘。在芯片的电极通道中插入四条铂丝, 使用一对镊子, 然后将其弯曲到塑料盘上, 以便在随后的步骤中对盘进行固定。插入导线 0.7-1 毫米入文化渠道, 通过 T 形渠道连接点. </li>
		<li> 在电极通道入口应用一滴 UV 固化胶水, 并允许胶水通过毛细管力在电极周围填充通道. </li>
		<li> 当它到达电极通道的末端时, 打开 UV 并固化胶水. 
		 警告: 不要看着紫外线光源, 因为这可能会伤害你的眼睛. </li>
		<li> 将四集成电极粘附在带有双组分环氧树脂胶粘剂的塑料盘上。这使得电极在测量过程中更容易处理, 而不会有从芯片中拔出电极的风险. </li>
		<li> 用透明胶片将芯片盖上, 然后在60和 #176 上烘烤; C 为 2 h. 允许冷却和储存无尘直到使用。这样, 它们可以安全地存储长达一个月. </li>
	</ol> </li> </ol> 2。脑源性内皮细胞的片内培养 <ol> <li> 对芯片进行涂膜以促进细胞附着。
	<ol> <li> 用磷酸盐缓冲盐水 (PBS) 填充两个通道, 在引入试剂之前将其浸湿。如果通道中有气泡, 请在显微镜下检查。如果是这样, 请使用额外的 PBS 冲洗删除它们. </li>
		<li> 用30和 #181 填充两个通道; 20 和 #181; PBS 中的人纤连蛋白。孵育在37和 #176; C 3 小时。检查气泡, 如果需要, 冲洗渠道与 PBS 和填充纤维连接蛋白溶液. </li>
		<li> 冲洗芯片与内皮生长培养基和孵化他们在37和 #176; C 和 5% CO 2 在孵化器为 2 h. </li>
		<li> 测量步骤3中描述的空白芯片的 TEER, 以确保所有电极与通道中的流体直接接触。在空芯片中, 典型的 TEER 值为0-1 和 #8486; cm 2 . </li>
	</ol> </li> <li> 将种子单元格放到顶部通道中。
	<ol> <li> 从培养瓶 (75 cm 2 区域性区域) 中移除培养液, 并与人脑微血管内皮细胞 (hCMEC/D3) 的汇合单层. </li>
		<li> 使用 PBS 清洗单元格。加入2毫升0.05% 胰蛋白酶-EDTA, 孵育在37和 #176; C 和 5% CO 2 2-5 分钟, 直到细胞从培养烧瓶中分离出来. </li>
		<li> 用培养基补充20% 胎牛血清 (FBS), 使胰蛋白酶停用。计数单元格并计算悬浮中的单元格总数. </li>
		<li> 同时, 离心的 hCMEC/D3 细胞在 390 x g 为5分钟, 并删除上清液. </li>
		<li> 重在适当体积的内皮生长培养基 (EGM-2) 中的细胞团, 以达到 5 x 10 6 细胞/毫升的浓度。这将导致芯片中的种子密度为 2 x 10 5 单元格/cm 2 。
		<li> 缓慢吸管30和 #181; L 混合细胞悬浮进入顶部通道, 并通过流畅的动作从入口移除吸管, 同时仍施加压力. </li>
		<li> 在显微镜下检查播种密度。在顶部通道中, 细胞的均匀分布应达到. </li>
		<li> 在37和 #176 上孵育芯片; C 和 5% CO 2 至少 1 h. 用内皮培养基冲洗任何附加细胞. </li>
	</ol> </li> <li> 保持芯片上的内皮细胞培养。
	<ol> <li> 每天两次, 插入区域性填充的吸管提示作为进气道中的储层, 并通过重力驱动流替换芯片内的介质. 
		注意: 为了避免气泡进入微和破坏细胞层, 确保在将针尖插入进进进进进进入口之前, 在吸管尖端和芯片顶部的流体之间进行流体接触。这可以通过在吸管插入前在/出水口添加一个小滴来方便. </li>
		<li> 还在插座中添加吸管端储液, 以防止通道干燥. </li>
		<li> 在37和 #176 上孵化芯片; C 和 5% CO 2 . </li>
	</ol> </li> </ol> 3。芯片 TEER 测量 <ol> <li> 设置 thTEER 测量装置, 包括锁相放大器和探头电缆电路, 如 Van der 头盔 et al 中所指定的那样。 16 或者, potentiostats 或阻抗分析器也适用于这些 impedance-based 的 TEER 测量. </li>
	<li> 从恒温箱中取出芯片, 使其达到室温至少10分钟. 从电极周围的塑料盘中取出任何液体, 以防止芯片外的电极桥接. </li>
	<li> 将两个电极的每个组合的阻抗谱从200赫兹到 1 MHz, 从而在每个芯片上产生6阻抗谱, 仅5-10 分钟. 检查阻抗谱是否具有预期的大小和形状以验证 TEER 测量, 如在结果部分中描述. </li>
	<li> 将芯片放回37和 #176 的恒温箱中; C 和 5% CO 2 完成测量后. </li>
	<li> 处理获得的阻抗谱以达到正常化的 TEER 值。
	<ol> <li> 获取测量的电阻 <img alt="Equation 1" src="/files/ftp_upload/56334/56334eq1.jpg"/> 之间的电极 <img alt="Equation 2" src="/files/ftp_upload/56334/56334eq2.jpg"/> 和<img alt="Equation 3" src="/files/ftp_upload/56334/56334eq3.jpg"/>, 如下所示 图 2B 和 D , 从电阻高原在对应的阻抗谱10赫. </li>
		<li> 计算 TEER 使用六测量的电阻对应于一个芯片在同一时间点使用的方程 图 2G 16 : 
		<img alt="Equation 20" src="/files/ftp_upload/56334/56334eq20.jpg"/> 在此等式中, <img alt="Equation 4" src="/files/ftp_upload/56334/56334eq4.jpg"/> 是测量电阻的区域性区域, 即 img alt = "方程式 5 "src ="/文件/ftp_upload/56334/56334eq5. jpg 是细胞屏障和细胞膜的阻力, <img alt="Equation 6" src="/files/ftp_upload/56334/56334eq6.jpg"/>,<img alt="Equation 7" src="/files/ftp_upload/56334/56334eq7.jpg"/>, <img alt="Equation 8" src="/files/ftp_upload/56334/56334eq8.jpg"/> 和 <img alt="Equation 9" src="/files/ftp_upload/56334/56334eq9.jpg"/> 是电阻通过细胞屏障和 <img alt="Equation 10" src="/files/ftp_upload/56334/56334eq10.jpg"/> 和 <img alt="Equation 11" src="/files/ftp_upload/56334/56334eq11.jpg"/> 测量的值是测量的电阻值直通道。 16 公式是从 图 2C 中所示的等效电路导出的。
	</ol> </li> <li> 在所有时间点从 TEER 中减去空白 TEER, 以及时获得 TEER 的开发. </li> </ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+the+blood-brain+barrier:+molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches.</a> Brain Res Rev. 64 (2), 328-363 (2010).</li><li>Weksler, B., 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=Blood-brain+barrier-specific+properties+of+a+human+adult+brain+endothelial+cell+line.">Blood-brain barrier-specific properties of a human adult brain endothelial cell line.</a> FASEB J. 19 (13), 1872-1874 (2005).</li><li>Chueh, B. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leakage-free+bonding+of+porous+membranes+into+layered+microfluidic+array+systems.">Leakage-free bonding of porous membranes into layered microfluidic array systems.</a> Anal chem. 79 (9), 3504-3508 (2007).</li><li>Golowasch, J., Nadim, F., Jaeger, D., Jung, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Computational Neuroscience. , 555-558 (2015).</li><li>Benson, K., Cramer, S., Galla, H. -. 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作者没有什么可透露的。

在本手稿中, 介绍了一种 organ-on-chip 装置的工程和直接测定在该装置中培养的细胞屏障的 transendothelial 电阻 (TEER)。提出了一种无尘室集成电极的方法, 采用四电极直接 TEER 测定法, 适用于具有两个微流控腔的 organ-on-chip 装置。只要在两个舱室中分离出四电极, 就可以调整芯片布局和几何形状, 以符合预想的实验要求。四电极甚至可以方便地插入到现有芯片的入口, 只要它们在六测量的持续时间内都是固定的。通过改变聚合物/甲苯比, 可以对不同的膜和通道的几何形状进行无泄漏键合的优化。一个更高的甲苯含量导致更薄的自旋涂层的砂浆层26 , 可能更适合在更浅, 更窄的渠道, 在硅橡胶零件。较低的甲苯含量会导致较厚的砂浆层26 , 更适合于将较厚的薄膜封装在该产品的部件之间。
正如在图 2A的示意性阻抗谱和图 2E 和 2F中的实验谱所示, 阻抗测量受电极介质界面上的双层电容的影响。由于微内电极的体积较小, 双层电容可以控制阻抗谱中细胞屏障的电阻高原, 使 TEER 的定量化复杂化。为了克服这一点 , 电极可以入到文化通道前固定。这将增加暴露在培养基中的电极的表面积, 而且双层电容也会增加。这导致电容性倾斜的转移到更低的频率, 因此细胞障碍的抵抗的高原可以更加容易地被认出和定量。虽然两个电极之间的测量路径的电阻会变小, 但这不会影响所提出方法的 TEER 量化。
在通过细胞屏障测量时, 可能会发现额外的电阻高原是不可能被识别的。这可能是两个通道之间的射流连接的结果, 例如, 如果薄膜被砂浆所包围, 就会导致在细胞屏障周围的测量路径。此外, 如果电极由培养基液滴连接, 则在芯片外部可以有电架桥。这通常是结合一个较低的测量阻抗, 可以解决通过消除这一桥梁的培养基。最后, 如果没有电阻高原和测量阻抗是数量级比预期的高, 可能有一个松散的连接在电线或电源。
在未来, BBB-on-chip 的生理相关性可以通过在生理水平上暴露内皮细胞的剪切应力来增加, 这被报道促进血脑屏障的分化和增加屏障的致密性, 很难达到在传统的体外模型中。29此外, 所提出的设备的底部通道提供了一个合适的隔室, 供大脑衍生细胞与内皮共。这也有望增加屏障功能, 也使研究的复杂互动之间的相关细胞类型的病理条件。29
最后, 本出版物中描述的 organ-on-chip 装置可以使用标准的实验室设备制作, 并显示为使用四集成电极的直接 TEER 测量, 不妨碍对所研究的细胞进行目视检查。层.通过模拟该芯片中的 BBB, 并在培养期内监测 TEER, 证明了该装置在上领域的适用性。其他的一些 (障碍) 器官可以被模仿, 包括其他相关的细胞类型进入这个芯片。此外, 测量 TEER 的方法可以很容易地应用在其他两个 compartment-based organ-on-chip 设备, 以达到可重复和有意义的 TEER 值, 可以通过设备和系统进行比较。

上是迅速涌现的新的和有为的类在体外组织模型。1在这些模型中, 细胞是在微流控设备中培养的, 它们的设计方法是模仿这些细胞的生理环境。1,2这将导致这些细胞的生理或病理行为比传统的体外模型的简单设计和基本功能更逼真。3,5,6此外, 上提供了比体内模型更好的可控环境, 并且可以将健康和病态的组织从人类起源中结合起来, 忠实地复制人类生理学和病理学。最近总结的发展的血脑屏障芯片 (BBBs-on-chips) 表明, 该领域是迅速向前迈进。7
上的另一个优点是, 通过显微镜、on-line 生化分析和集成传感器, 它们能够 real-time 和连续监测设备内培养的组织。1,2例如, 测量 transendothelial 或 transepithelial 电阻 (TEER) 是一种强有力的方法, 用于监测性障碍形成组织的发育和破坏。8,9,10 TEER 是跨细胞屏障的电阻, 因此表明了屏障的完整性和渗透性。10在上中, 细胞屏障通常在分离两个流体通道的膜上培养, 代表该屏障组织的顶端和侧隔间。在这种芯片中, TEER 测量可以方便地进行, 电极插入到两个通道的入口和出口。3,4,11,12,13,14,15但是, 手动插入和重新加入电极可以很容易地导致放置错误, 从而在测量电阻的变化, 如例如通过微的长期或较短路径的阻力的差异与细胞屏障阻力相比有显著性差异。16为了消除重新插入的错误, 提出了集成电极的器件。然而, 大多数这些集成电极在检查组织培养时阻止视图17,18,19,20,21和/或需要专门洁净室工艺制造。17,22
本出版物中介绍的 organ-on-chip 设备, 首先应用于早期出版物,16将集成电极的稳定性与测量的电池层的可见性结合起来, 并易于制作。该芯片的设计和制作在图 1中进行了描述。总之, 该装置由两个烷 (聚碳酸酯) 部分与通道印记, 是粘合在一起的无泄漏与0.4 µm 孔在两者之间。四白金线电极插入和固定到位与固化胶粘剂井在文化区域外面。所有这些制造步骤都可以使用一般的实验室设备进行, 而不需要一个洁净室环境。最重要的是, 六阻抗测量可以使用这四电极, 从而允许直接隔离的测量 TEER, 独立的电阻的微领先的横断面, 从而尽量减少影响系统中的非生物变体, 如 (重新) 插入错误。16
为了证明这个装置的适用性和直接 TEER 测量, 血脑屏障 (BBB) 被复制在这个芯片。这种生物屏障由专门的内皮细胞组成, 调节血液和大脑之间的运输, 以提供大脑的稳态。23,24为了模拟 BBB, 微流控装置的顶部通道内衬有人脑显微血管内皮细胞, 从 hCMEC/D3 细胞系 (Dr. P-O 提供。Couraud, INSERM, 巴黎, 法国)。25然而, 所提出的方法更普遍地适用于任何具有两个舱室的 organ-on-chip 设备, 从而能够使用易于集成的电极直接 TEER 测定。
本文首先介绍了集成电极 organ-on-chip 装置的制作过程。其次, 对该装置内的脑内皮细胞的播种过程和培养进行了解释, 以及对芯片 TEER 的测量。在结果部分中, 显示了具有代表性的 TEER 测量, 并对数据处理进行了澄清。最后, 对 BBB-on-chip 的屏障功能进行了3天的监测, 表明了所提出的装置和方法对 TEER 的适用性。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) base agent and curing agent: Sylgard 184 Silicone elastomer kit</td><td>Dow Corning</td><td>1673921</td><td></td></tr><tr><td>Scotch Magic tape</td><td>3M</td><td></td><td></td></tr><tr><td>Biopsy punch, 1.0 mm diameter</td><td>Integra Miltex</td><td>33-31AA-P/25</td><td></td></tr><tr><td>Polycarbonate membrane, 0.4 &amp;micro;m pore size</td><td>Corning</td><td>3401</td><td>Cut from Transwell culture inserts</td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>244511</td><td></td></tr><tr><td>Platinum wire, 200 &amp;micro;m diameter</td><td>Alfa Aesar</td><td>10287</td><td></td></tr><tr><td>UV-curable adhesive: Norland Optical Adhesive 81</td><td>Norland Products</td><td>NOA 81</td><td></td></tr><tr><td>Epoxy adhesive: Loctite M-31 CL Hysol</td><td>Henkel</td><td>30673</td><td></td></tr><tr><td>hCMEC/D3 cells</td><td></td><td></td><td>Human cerebral microvascular endothelial cell line, kindly provided by Dr. P.-O. Couraud, INSERM, Paris, France</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Human plasma fibronectin, 20 &amp;micro;g/ml</td><td>Gibco</td><td>33016015</td><td></td></tr><tr><td>Endothelial growth medium-2 (EGM-2)</td><td>Lonza</td><td>CC-3162</td><td>Endothelial basal medium-2 (EBM-2) supplemented with EGM-2 SingleQuots</td></tr><tr><td>Trypsin-EDTA, 0.05%</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oven</td><td>Binder</td><td>9010-0190</td><td></td></tr><tr><td>Spin coater: Spin 150</td><td>Polos</td><td>SPIN150-NPP</td><td></td></tr><tr><td>UV light source, 365 nm for 5 s at 350 mW/cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Manufactured in-house</td><td></td><td></td></tr><tr><td>Centrifuge: Allegra X-12R Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Incubator</td><td>Binder</td><td>CB E2 150</td><td></td></tr><tr><td>Boxense</td><td>LocSense</td><td></td><td>Lock-in amplifier with probe cable circuit, specialized for on-chip TEER measurements</td></tr></tbody></table>

fabrication and validation of an organ-on-chip system with integrated electrodes to directly quantify transendothelial electrical resistance

上,体外模型涉及微流控装置内 (人体) 组织的培养, 正在迅速涌现, 并有望为研究人类健康和疾病提供有用的研究工具。为了表征 organ-on-chip 装置内培养的细胞层的屏障功能, 通常 transendothelial 或 transepithelial 电阻 (TEER) 进行测量。为此, 电极通常集成到芯片的微加工方法, 以提供更稳定的测量比通过手工插入电极进入芯片的入口。然而, 这些电极经常妨碍对所研究的细胞层进行目视检查, 或需要昂贵的洁净室工艺进行制造。为了克服这些限制, 这里所描述的设备包含了四容易集成的电极, 它们被放置在文化区域之外并进行固定, 使目视检查成为可能。使用这四电极六测量路径的抵抗可以是定量的, TEER 可以直接地被隔绝, 独立抵抗文化中等填装的微。在该装置中复制了血脑屏障, 并对其 TEER 进行了监测, 以表明该装置的适用性。这个芯片, 集成电极和 TEER 测定方法一般适用于上, 既模仿其他器官或被纳入现有的 organ-on-chip 系统。

在图 2A中显示了通过无单元 (实线) 和通过蜂窝屏障 (虚线) 的芯片进行电阻抗谱的示意图结果。四主要区域可以被辨认, 每个由一个具体电组分控制。在大约1赫之下, 双重层数电容在电极-文化媒介接口控制, 描绘的是以消极倾斜为阻抗大小和相移接近-90 °。双层电容占主导地位的频率, 取决于暴露在培养基中的电极面积。电阻高原高于1赫 (无细胞) 或100赫 (与细胞) 相移接近0°, 对应于培养基的阻力在微流控通道内, 取决于通道长度和横截面积, 并在膜, 取决于孔隙度和厚度。当测量通过一个细胞屏障, 一个额外的电阻高原之间的1和10赫被视为以及一个局部最大的相图。这个区域对 TEER 的确定至关重要, 因为当细胞存在于测量的路径中时, 阻抗的结果明显增加, 因此被称为 "感兴趣的区域"。额外倾斜在10和100赫之间对应于细胞阻挡电容, 从电绝缘脂质双层膜出现并且取决于细胞层数的总面积。27这些区域的边界以及阻抗大小取决于正在研究和改变的系统, 其中包括通道尺寸、培养基电导率、电极位置和细胞类型。为进一步阅读障碍形成组织的电阻抗谱理论和实践推荐本森et al.的评论文章。28
TEER 测量的代表性数据分别显示在图 2E 和 2F中的空白芯片和带有 hCMEC/D3 脑血管内皮细胞层的芯片。简而言之, 阻抗光谱学使用六测量与四电极: 二测量通过细胞培养中等填充的渠道 (坚实线) 和四测量通过渠道并且膜并且-如果存在-蜂窝屏障 (虚线)。这些六测量路径可以在图 2B的等效电阻电路中进行识别, 它是从示意图剖面 (图 2C) 和顶部视图 (图 2D) 派生的。空白芯片测量 (图 2E) 显示了不带单元格的阻抗谱的典型形状, 如图 2A所示。通过蜂窝屏障 (图 2F中的虚线) 的测量类似于图 2A中带有单元格的典型阻抗谱。注意, 阻抗的大小和相移增加到1兆赫。这是高频率测量设置的典型响应, 不是实验源。
利用这些实验阻抗谱确定 TEER, 首先确定了测得的芯片电阻, 即细胞层、通道和膜的总电阻。为此, 选择一个适当的读出频率在感兴趣区域, 是在地方最大在阶段剧情与细胞和接近0°相移没有细胞:10 赫。以六测量的抵抗在10赫, 测量在四电极之间, TEER 用等式直接地计算在图 2F。
为了表明该装置适用于上中的 TEER, 血脑屏障在芯片内被复制, 并在3天的培养过程中对其 TEER 进行了监测。在图 3A中, 平均值 (SEM) 的平均 TEER ±标准误差显示为四 BBBs-on-chips, 导致22±1.3 Ω cm2的高原, 这与在文献中报告的该细胞系的 TEER 相媲美。29此外, 在实验结束和细胞核的荧光染色后, 可以看到, 大脑内皮在设备内形成了一个连续的单层 (图 3B)。紧密结合蛋白紧密 Occludens-1 (ZO-1) 的免疫荧光染色表明, 脑内皮细胞维持其 BBB 特异性表型, 形成紧密的连接配合物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56334/56334fig1.jpg"> 
图 1: 集成电极的 organ-on-chip 装置的设计和装配。 
(A)微流控芯片的分解视图, 由顶部通道 (TC)、膜 (M) 和底部通道 (BC) 的底部的硅橡胶部分组成。四白金线电极 ( E1 - 4 ) 入并且固定在旁边渠道。在顶部通道和膜顶部, hCMEC/D3 脑内皮细胞被培养, 以复制 BBB。(B)组装好的芯片, 固定在塑料盘上。转载和改编的许可, 从唯。16 <a href="//ecsource.jove.com/files/ftp_upload/56334/56334fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56334/56334fig2.jpg"> 
图 2: 有代表性的阻抗数据和 TEER 的测定。 
(A)示意图阻抗谱显示阻抗大小 (Ω) 和相移 (°) 与频率 (Hz), 典型的电阻抗谱在芯片上没有细胞 (实线) 和与细胞 (虚线)。主要有四个区域, 各占主导地位: 电极上的双层电容、培养基电阻、细胞阻挡电阻和细胞膜电容。"感兴趣的区域" 表示细胞层的贡献可以量化 (红色箭头)。(B)芯片的等效电阻电路, 显示顶部通道电阻器 r1和 r3、底部通道电阻 r2和 r4和膜和 EC 阻挡电阻 rm。(C)图示剖面, 显示在顶部通道中培养的内皮细胞 (EC)。(D) "BBB 芯片" 的示意图显示了电极的配置以及 0.25 mm2的区域性, 通过它来测量阻抗。(E)填充有细胞培养基的空白芯片的代表性阻抗谱。(F)具有代表性的 hCMEC/D3 脑内皮细胞在3天内培养的芯片的阻抗谱。(G)公式从四电极的所有六组合之间的测量电阻中计算 TEER。转载和改编的许可, 从唯。16 <a href="//ecsource.jove.com/files/ftp_upload/56334/56334fig2large.jpg" target="_blank">请单击此处查看较大的限制</a>这个数字的离子。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56334/56334fig3.jpg"> 
图 3: BBB-on-chip 的代表性 TEER 发展。 
(a)平均 TEER ±标准误差 (sem) 四 BBBs-on-chips 在三天的文化期间, 到达一个高原在22±1.3 Ω cm2 (平均± sem)。为比较, 空白芯片的数据包括, 显示的边际变化和偏差从0Ω cm2在同一期间与芯片的变动和 TEER 价值相比与细胞。(B)染色细胞核的荧光显微镜显示, 在嵌入的位置上, 有一个连续单层的内皮细胞。(C)免疫荧光分析显示致密结蛋白紧密 Occludens-1 的存在, 表明细胞之间的 BBB 特定的紧密连接引起了测量的 TEER。转载和改编的许可, 从唯。16 <a href="//ecsource.jove.com/files/ftp_upload/56334/56334fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance at JoVE.com

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

这本出版物描述了一个 organ-on-chip 装置的制造与集成电极直接量化的 transendothelial 电阻 (TEER)。为验证, 在该微流控装置内模拟血脑屏障, 监测其屏障功能。所提出的电极集成方法和直接 TEER 定量是普遍适用的。

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

fabrication-validation-an-organ-on-chip-system-with-integrated

Research

JoVE Journal

Bioengineering

16.2K 次观看.  University of Twente. 这本出版物描述了一个 organ-on-chip 装置的制造与集成电极直接量化的 transendothelial 电阻 (TEER)。为验证, 在该微流控装置内模拟血脑屏障, 监测其屏障功能。所提出的电极集成方法和直接 TEER 定量是普遍适用的。

视频: Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

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 ...

科研

生物工程

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

发育生物学

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

<meta charset="utf8"></head><body>用于 A431 细胞电穿孔分析和递送预测的 TEEI 方法

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

<meta charset="utf8"></head><body>作者聚焦：通过微通道和纳米通道优化多孔衬底电穿孔，以增强监测和中间阶段表征

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

基于多孔底物的电穿孔与跨上皮电阻抗监测

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

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

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

摘要

探索更多视频

此视频中的章节

阻隔组织破坏与有机电化学晶体管传感

生成基于人类 iPSC 的血脑屏障芯片

可伸缩、双通道、微流控芯片制造

具有透化检测功能的连续流动微电穿孔系统的制造和操作

结合人体类器官和芯片器官技术，对肠道区域特异性功能进行建模

在开顶器官芯片上重建人上皮组织的细胞结构和功能

基于多孔底物的电穿孔与跨上皮电阻抗监测

基于多孔底物的电穿孔与跨上皮电阻抗监测

基于多孔底物的电穿孔与跨上皮电阻抗监测

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