Abstract：
Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.
摘要：
3D人表皮等效物（HEE）是临床前研究皮肤病学和调节毒理学中最先进的器官型培养模型。这里,我们研究了电阻抗谱（EIS）用于非侵入性测量HEE表皮屏障功能的实用性。我们的设置包括定制的盖子，该盖子具有在标准24-transwell细胞培养系统上对齐的12个电极对。连续7天的连续EIS测量不影响表皮形态，读数显示出与仅测量一次的HEE相当的趋势。我们在所得阻抗谱中确定了两个频率范围：称为EISdiff的较低频率范围与角质形成细胞终末分化相关，而与表皮厚度无关，称为EISSC的较高频率范围与角质层厚度相关。从CRISPR/Cas9工程角质形成细胞产生的HEE，缺乏关键分化基因FLG，TFAP2A,AHR或CLDN1证实角质形成细胞终末分化是定义EISdiff的主要参数。暴露于促炎性银屑病或特应性皮炎相关的细胞因子混合物降低了角质形成细胞分化标志物的表达并降低了EISdiff。这种细胞因子相关的EISdiff降低在用治疗性分子刺激后正常化。总之,EIS提供了一种非侵入性系统，可以连续和定量地评估HEE屏障功能，并灵敏和客观地测量屏障的发展。缺陷和修复。
