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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In vitro reconstructed skin models have been widely used to study epidermal physiology. However, these models rely on primary cells that are limited by availability and donor variations. To overcome these issues we validated immortalized cell lines that were generated by the expression of SV40 large T antigen and hTERT (NHEK-SV/TERT) or HPV E6/E7 (NHEK-E6/ E7). Both cell lines show indefinite growth up to over 50 passages and displayed increased growth rates compared to primary cells. Primary keratinocytes present a mix of different epidermal cell populations such as keratinocyte stem and cells already committed to differentiation. Immortalized cells did not show distinct populations, arguing that immortalized cells are a more uniform mainly proliferative cell population that needs an exogenous stimulus to commit to differentiation. While NHEK-E6/E7 show a differentiation behavior comparable to primary cells, the highly proliferative NHEK-SV/TERT needed withdrawal of any proliferative stimulus to commit to a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and formed a tight and functional barrier. Both cell lines responded to inflammatory stimuli leading to an epidermal morphology mimicking Th1 or Th2 type dermatosis. In addition these cell lines are suitable for genome editing using CRISPR/Cas9 technology as different target genes could be successfully knocked out with NHEK-SV/TERT showing a higher transfection efficiency simplifying the isolation of edited single cell clones. Thus, the tested cell lines are suitable substitutes for primary cells and have the potential to serve as suitable models to investigate molecular pathomechanisms and test novel therapeutics during pre-clinical evaluation.