Abstract：
Measurement of endothelial and epithelial barrier integrity is important for a variety of in vitro models, including Transwell assays, cocultures, and organ-on-chip platforms. Barrier resistance is typically measured by trans-endothelial electrical resistance (TEER), but TEER is invasive and cannot accurately measure isolated monolayer resistance in coculture or most organ-on-chip devices. These limitations are addressed by porous membrane electrical cell-substrate impedance sensing (PM-ECIS), which measures barrier integrity in cell monolayers grown directly on permeable membranes patterned with electrodes. Here, we advanced the design and utility of PM-ECIS by investigating its sensitivity to working electrode size and correlation with TEER. Gold electrodes were fabricated on porous membrane inserts using hot embossing and UV lithography, with working electrode diameters of 250, 500, and 750 μm within the same insert. Sensitivity to resistance changes (4 kHz) during endothelial barrier formation was inversely proportional to electrode size, with the smallest being the most sensitive (p < 0.001). Similarly, smaller electrodes were most sensitive to changes in impedance (40 kHz) corresponding to cell spreading and proliferation (p < 0.001). Barrier disruption with both EGTA and thrombin was detectable by all electrode sizes. Resistances measured by PM-ECIS vs TEER for sodium chloride solutions were positively and significantly correlated for all electrode sizes (r > 0.9; p < 0.0001), but only with 750 μm electrodes for endothelial monolayers (r = 0.71; p = 0.058). These data inform the design and selection of PM-ECIS electrodes for specific applications and support PM-ECIS as a promising alternative to conventional TEER for direct, noninvasive, real-time assessment of cells cultured on porous membranes in conventional and organ-on-chip barrier models.
摘要：
内皮和上皮屏障完整性的测量对于多种体外模型是重要的,包括Transwell分析,cocultures,和器官芯片平台。屏障阻力通常通过跨内皮电阻(TEER)来测量，但是TEER是侵入性的，不能准确测量共培养或大多数器官芯片设备中孤立的单层电阻。这些限制通过多孔膜电池-基底阻抗传感(PM-ECIS)解决，它测量直接在用电极图案化的可渗透膜上生长的细胞单层中的屏障完整性。这里,我们通过研究PM-ECIS对工作电极尺寸的敏感性以及与TEER的相关性，提高了PM-ECIS的设计和实用性。使用热压花和UV光刻技术在多孔膜插入物上制造金电极，工作电极直径为250、500和750μm的同一插入件。内皮屏障形成过程中对电阻变化（4kHz）的敏感性与电极大小成反比，最小的是最敏感的(p<0.001)。同样，较小的电极对与细胞扩散和增殖相对应的阻抗变化(40kHz)最敏感(p<0.001).所有电极尺寸均可检测到EGTA和凝血酶的屏障破坏。对于所有电极尺寸（r>0.9；p<0.0001），通过PM-ECIS与TEER对氯化钠溶液测得的电阻呈正相关且显着相关。但只有750μm电极用于内皮单层（r=0.71；p=0.058）。这些数据为特定应用的PM-ECIS电极的设计和选择提供了信息，并支持PM-ECIS作为传统TEER的有希望的替代产品，非侵入性，在常规和器官芯片屏障模型中，实时评估在多孔膜上培养的细胞。
