Klaka Patricia, Grüdl Sabine, Banowski Bernhard, Giesen Melanie, Sättler Andrea, Proksch Peter, Welss Thomas, Förster Thomas
Henkel AG & Co. KGaA, Düsseldorf, Germany.
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University, Düsseldorf, Germany.
PLoS One. 2017 Aug 10;12(8):e0182752. doi: 10.1371/journal.pone.0182752. eCollection 2017.
Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.
人类外分泌汗腺功能失调会对患有多汗症等疾病的人的生活质量产生负面影响。无法出汗甚至会对患有无汗症的人造成严重的健康影响。必须阐明其潜在机制，并开发一种可靠的体外药物筛选测试系统。在此，我们描述了一种由原代人外分泌汗腺细胞制成的新型器官型三维（3D）汗腺模型。初步实验表明，二维（2D）培养的外分泌汗腺细胞会失去典型的生理标志物。为了尽可能接近体内情况，我们应用了悬滴培养技术，当在其自然球形环境中培养时，大部分标志物得以恢复。为了比较器官型3D汗腺模型与体内人类汗腺，我们使用转录组学和蛋白质组学分析比较了与外分泌汗腺相关的标志物。比较标志物谱，显示出高度的体外-体内相关性。在3D模型中发现癌胚抗原相关细胞粘附分子5（CEACAM5）、毒蕈碱型乙酰胆碱受体M3（CHRM3）、钠-钾-氯共转运体1（NKCC1）、钙激活氯通道anoctamin-1（ANO1/TMEM16A）和水通道蛋白5（AQP5）有显著表达水平。此外，用乙酰胆碱或毛果芸香碱进行胆碱能刺激会导致在钙流测定中监测到钙内流。用作汗腺模型系统的汗腺细胞系NCL-SG3无法实现胆碱能刺激。我们的结果表明，在必需的外分泌汗腺关键调节因子的表达以及对刺激的生理反应方面，器官型3D汗腺模型相对于2D培养具有明显优势。综上所述，这种新型器官型3D汗腺模型显示出良好的体外-体内相关性，是筛选调节出汗机制的潜在生物活性物质的合适替代物。
