In the fields of tissue engineering and drug discovery, confirming the formation and maturation of epithelial cell tight junctions (TJs), which are necessary for blocking pathogenic invasion and absorption of nutrients and ions, at a high spatiotemporal resolution is essential. We previously developed a system of monitoring pH perturbation induced by weak acid exposure to cells cultured on an ion-sensitive field-effect transistor that enables a sensitive and specific detection of biomembrane injuries and TJ breakdowns caused by external stimuli such as nanomaterials and cytotoxins. In this study, we monitor time-lapse changes in the paracellular diffusion of growing epithelial cell monolayers using the pH perturbation assay as well as conventional permeability and trans-epithelial electrical resistance assays. The effects of the extracellular matrix and a TJ potentiator (KN-93) on epithelial TJ formation are evaluated. TJ formations were promoted on the substrate coated with Matrigel more than on the one coated with poly(l-lysine). KN-93 accelerated TJ formations in a dose-dependent manner. The pH perturbation assay denoted a longer incubation time for the completion of TJ formation compared with the conventional assays under the same conditions. Importantly, the pH perturbation assay is able to rigorously evaluate TJ formation, as the assay uses protons as the smallest indicator for detecting paracellular gaps, and the pH perturbation is specific to TJ alterations. These features for in vitro TJ evaluation using proton dynamics are advantageous for applications in tissue engineering and drug development.

中文翻译：

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半导体表面上的感应质子动力学，用于感测细胞外基质和药物增强的紧密结形成。

在组织工程和药物发现领域，确认上皮细胞紧密连接（TJs）的形成和成熟对阻止病原菌入侵和吸收养分和离子具有很高的时空分辨率，这是必不可少的。我们以前开发了一种监测pH扰动的系统，该pH扰动是由弱酸暴露于离子敏感的场效应晶体管上培养的细胞引起的，该系统能够灵敏且特异地检测由外部刺激（例如纳米材料和细胞毒素）引起的生物膜损伤和TJ击穿。在这项研究中，我们使用pH扰动测定法以及常规的通透性和跨上皮电阻测定法监测生长中的上皮细胞单层的副细胞扩散随时间的变化。评估了细胞外基质和TJ增强剂（KN-93）对上皮TJ形成的影响。与涂有聚（1-赖氨酸）的基质相比，涂有Matrigel的基质对TJ的促进作用更大。KN-93以剂量依赖性方式加速TJ的形成。与在相同条件下的常规测定相比，pH扰动测定表明完成TJ形成的孵育时间更长。重要的是，pH扰动测定能够严格评估TJ的形成，因为该测定使用质子作为检测细胞旁间隙的最小指示剂，并且pH扰动特定于TJ改变。这些使用质子动力学进行体外TJ评估的功能，在组织工程和药物开发中具有优势。