Electrical impedance spectroscopy in human epidermal equivalents

Abstract

3 D human epidermal equivalents (HEEs) are state‐of‐the‐art organotypic culture models used in investigative dermatology yet they lack accurate, reliable and easy to use techniques to measure skin barrier function. Here, we investigated the utility of electrical impedance spectroscopy (EIS) for non–invasive 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. Before measurements, HEEs were acclimated to room temperature and cultures were lowered to the middle position in the transwell plate while 1600 μL PBS at room temperature was added below and 500 μL PBS on top of the filter. Thereafter, the smart lid was placed on the wells ensuring both electrodes being submerged. Following device self–calibration, impedance was measured over a frequency range from 10 Hz to 100,000 Hz in 30 logarithmic intervals. Measurement output contains impedance as well as phase values. Phase values can be interpreted as is while a PBS only blank measurement was subtracted from the corresponding electrode of the impedance output. We determined two frequency ranges in the resulting impedance spectra. For EISdiff (127–2212 Hz) and EIS SC (28,072–100,000 Hz) the area under the curve was calculated using the respective frequency ranges. EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and EIS SC correlated with stratum corneum thickness. Serial EIS measurements for seven consecutive days did not impact epidermal morphology and readouts showed comparable trends to HEEs measured only once. 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 pro–inflammatory 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 non–invasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects and repair.