Investigation on Ciliary Functionality of Different Airway Epithelial Cell Lines in Three-Dimensional Cell Culture

Three-dimensional respiratory tissue models have been generated using, for example, human primary airway epithelial cells (hAEC) or respective cell lines. To investigate ciliopathies, such as primary ciliary dyskinesia, the presence of functional kinocilia in vitro is an essential prerequisite. Since access to hAEC of healthy donors is limited, we aimed to identify a respiratory epithelial cell line that is capable to display functional kinocilia on at least 60% of the apical surface. Thus, we cultured four different human respiratory cell lines with human primary airway fibroblasts under airlift conditions, characterized the morphology, and analyzed ciliary function. Only one of the tested cell lines showed beating kinocilia; however, <10% of the whole surface was covered and ciliary beating was undirected. Positive control tissue models using hAEC and fibroblasts displayed expected directed ciliary beating pattern around 11 Hz. Our data show that the available cell lines are not suitable for basic and applied research questions whenever functional kinocilia are required and that, rather, hAEC- or human induced pluripotent stem cell-derived tissue models need to be generated. Impact Statement To study ciliopathies or Bordetella pertussis infection in vitro, three-dimensional respiratory tissue models with functional kinocilia covering at least 60% of the model's surface are mandatory. We cultured four respiratory cell lines on a fibroblast-loaded biological scaffold and showed that none of them met this requirement. In contrast, primary airway cell-derived models sufficiently reflected the mucociliary phenotype. To further search for an alternative to primary respiratory cells, investigations on other cell lines should be conducted or even new cell lines have to be generated.