Abstract. Skin fulfils a plethora of eminent physiological functions ranging from physical barrier over immunity shield to the interface mediating social interaction. Prone to several acquired and inherited diseases, skin is therefore a major target of pharmaceutical and cosmetic research. The lack of similarity between human and animal skin and rising ethical concerns in the use of animal models have driven the search for novel realistic three-dimensional skin models. This review provides a survey of contemporary skin models and compares them in terms of applicability, reliability, cost and complexity.Keywords: Skin, phenotypic screening, 3D models, pharmaceutical research Skin -composition and functionHuman skin covers an area of almost 2 m 2 in the adult and consists of the three major layers, subcutis, dermis, and epidermis. The subcutis is composed of adipose and epithelial cells. It harbours blood vessels, neurites of peripheral neurons, Vater-Pacini mechanosensors, and, partially, also sweat glands and hair follicles. It connects the skin to periosteum and fascia, absorbs forces, and mediates thermal insulation. The dermis supplies the epidermis with mechanical support and nutrients. It is stratified into an inner, reticular, and an outer, papillary, zone. The dermis houses most sebaceous glands, sweat glands, hair follicles, smooth muscle cells, and capillary beds and, thus, regulates skin moisture, body temperature, and performs the secretory function of skin. The papillary layer is characterized by relatively loose connective tissue, where Meissner corpuscles sense touch. Immune cells, particularly mast cells and dendritic cells, are patrolling in the papillary layer and mediate local inflammatory reactions and immune surveillance. Finally, dermal fibroblasts secrete extracellular matrix (ECM) and basement membrane components. These are primarily collagens I and III, and a proteoglycan-rich ground substance [1]. The resulting ECM mediates tensile strength of the dermis. The dermo-epidermal junction is centered around a special basement membrane. This is composed of a laminin/collagen IV scaffold and further typical basement membrane components such as perlecans and nidogens [1].The epidermis is a squamous epithelium of 50-100 m thickness. It is devoid of blood vessels but contains keratinocytes, Merkel cell mechanosensors, Langerhans immune cells, and melanocytes. The 1 These authors contributed equally.

Sweating is an important physiological process to regulate body temperature in humans, and various disorders are associated with dysregulated sweat formation. Primary sweat secretion in human eccrine sweat glands involves Ca(2+) -activated Cl(-) channels (CaCC). Recently, members of the TMEM16 family were identified as CaCCs in various secretory epithelia; however, their molecular identity in sweat glands remained elusive. Here, we investigated the function of TMEM16A in sweat glands. Gene expression analysis revealed that TMEM16A is expressed in human NCL-SG3 sweat gland cells as well as in isolated human eccrine sweat gland biopsy samples. Sweat gland cells express several previously described TMEM16A splice variants, as well as one novel splice variant, TMEM16A(acΔe3) lacking the TMEM16A-dimerization domain. Chloride flux assays using halide-sensitive YFP revealed that TMEM16A is functionally involved in Ca(2+) -dependent Cl(-) secretion in NCL-SG3 cells. Recombinant expression in NCL-SG3 cells showed that TMEM16A(acΔe3) is forming a functional CaCC, with basal and Ca(2+) -activated Cl(-) permeability distinct from canonical TMEM16A(ac). Our results suggest that various TMEM16A isoforms contribute to sweat gland-specific Cl(-) secretion providing opportunities to develop sweat gland-specific therapeutics for treatment of sweating disorders.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid-and chip-based threedimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-tonoise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols-in particular, for screening purposes-clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume‐regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT‐ and NHEK‐LRRC8A knockout cells by using CRISPR‐Cas9. I− influx assays using halide‐sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein‐AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.

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