O21 Development of a functional 3D eccrine sweat gland model

Abstract Introduction and aims Human skin is populated by a uniquely high density of eccrine sweat glands (ESGs), which are primarily responsible for thermoregulation. Eccrine sweat also contains moisturizing factors and antimicrobial peptides, which contribute to maintaining a healthy skin barrier. To date, the absence of functional, nonanimal models to study the human ESG is a significant challenge to researchers. This project aimed to produce a functional, microfluidic ESG-on-chip. Methods Microfluidic polydimethylsiloxane chips were fabricated via soft lithography, comprising three parallel channels separated by microposts, bonded to a glass coverslip. The central hydrogel-filled channel mimicked dermal ECM, with two parallel channels on either side for the ESG tubule and media perfusion, respectively. EC23 is a novel human ESG cell-line, which formed a confluent tubule within 24 h of microfluidic culture. Retention of fluorescein isothiocyanate-dextran in the lumen of the cell-lined channel confirmed the establishment of an epithelial barrier. Results The ESG-on-chip expressed proteins critical to secretory function, including muscarinic receptor 3 (M3), aquaporin 5, Na-K-Cl cotransporter 1 (NKCC1), anoctamin-1 (ANO1) and stromal interaction molecule 1 (STIM1) (n = 3). Analysis of calcium flux in the ESG-on-chip was also investigated using intracellular calcium indicator Fluo8. Stimulating with the M3 agonist carbachol increased intracellular calcium in a dose-dependent manner (n = 3). Additionally, an intracellular pH (pHic) assay demonstrated NKCC1-mediated ion transport by examining pHic recovery after treatment with ammonium chloride. The rate of pHic recovery (ΔpH/second) increased following pretreatment with carbachol, which stimulates NKCC1 activity via an increase in intracellular calcium. Conversely this response was blocked by the specific NKCC1 inhibitor bumetanide (n = 4). Conclusions This ESG-on-chip model replicates key structural, molecular and functional aspects of the human ESG. The ESG model and quantitative assays established here represent new experimental tools for assessing human ESG functionality in response to drugs, chemicals or environmental changes in vitro.