Abstract
In the preclinical testing of novel drugs cell-based in vitro models can be used to replace animal testing. The cell line CI-pNaEC is a promising candidate for a suitable in vitro model of the nasal epithelium but suffers from low ciliation and inconsistent epithelial architecture. In this study, the influence of Notch and ROCK inhibition on differentiation benchmarks in CI-pNaEC was investigated. Ciliation was monitored via immunofluorescence for acetylated α-tubulin, mucus expression was visualized and quantified using alcian blue, barrier properties were assessed using TEER measurements and permeation studies, while histology was evaluated by light microscopy. Treatment with low-micromolar concentrations of DAPT markedly increased the proportion of ciliated cells and promoted a more uniform, pseudostratified epithelial layer, while mucus secretion remained comparable to untreated controls. However, DAPT also induced a slight increase in barrier properties, resulting in permeation coefficients below ex vivo nasal tissue benchmarks for some markers. In contrast, Y-27632 alone had a visible effect on ciliation and histological uniformity, with minimal effects on mucus expression, and its combination with DAPT did not further improve differentiation. The results demonstrate that targeted Notch inhibition at low doses significantly enhances ciliation and epithelial organization in CI-pNaEC without compromising mucin production, although barrier function is modestly affected.