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

Abstract

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.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 fibroblastloaded 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. IntroductionT hree-dimensional (3D) cell cultures of the respiratory epithelium/mucosa afford research on-for examplethe impact of airborne pollutants, host-pathogen interactions, and drug permeation. 1-5 As source for respiratory epithelial cells, primary cells, induced pluripotent stem cells (iPSC), or cell lines can be used. Both primary cells and iPSC provide the opportunity to generate personalized tissue models, for example, to study individual drug responses or drug efficacy. Moreover, they show a high in vitro-in vivo correlation. These models feature a pseudostratified epithelial morphology, barrier properties, basal cells, mucus-producing goblet cells, and ciliated cells facilitating mucociliary clearance. [6][7][8][9]

Susceptibility of primary human airway epithelial cells toBordetella pertussisadenylate cyclase toxin in two- and three-dimensional culture conditions

“…It was previously found that after silica nanoparticle exposition, HBEC3-KT secreted significantly lower concentrations of IL-6 compared to the respiratory cell line BEAS-2B. 24 We found in our previous study 9 that another part of the innate immune system, the differentiation to the mucociliary phenotype, was impaired in HBEC3-KT. Thus, we assume that HBEC3-KT are not suitable to study the impact of CyaA further, since the innate immune response, at least in terms of IL-6 secretion and mucociliary differentiation, differs from observations made in primary respiratory epithelial cells.…”

“…As a 3D scaffold for tissue model generation, we used decellularized segments of the porcine small intestinal submucosa (SIS), as described elsewhere. 9,14,15 Animal research was performed according to the German law and institutional guidelines approved by the Ethics Committee of the District of Unterfranken, Wu¨rzburg, Germany (approval number: 55.2-2532-2-256). SIS segments were fixed between two cylinders.…”

“…However, several airway epithelial cell lines display only certain features of the corresponding primary cells. 8,9 To our knowledge, the impact of CyaA on primary human airway epithelial cells (hAEC) has not yet been investigated. Challenges while using primary hAEC are that they are difficult to standardise and establish in large quantities due to complex culture techniques, shortness of donor cells, low passaging capability 10 and a high potential of variability between donors.…”

RSV and HMPV Infections in 3D Tissue Cultures: Mechanisms Involved in Virus-Host and Virus-Virus Interactions

“…Limitations of the in vivo (mainly linked to animal experimentation and species specificity) and in vitro (especially morphological and functional variations in non-differentiated versus differentiated cells, respectively) models were overcome thanks to the use of three-dimensional in vitro tissues reconstituted from primary cells. Such ex vivo models are morphologically and functionally close to the airway epithelium and constitute the most suitable approach to study human viral respiratory infections [ 21 , 22 ]. During the 21st century, more 3D culture models, such as rotating wall vessel (RWV) and NASA 3D systems, have also been extensively used as an appropriate surrogate model to recapitulate the 3D architecture and the multicellular complexity of tissues observed in vivo and study virus-host interaction including RSV [ 23 , 24 , 25 , 26 , 27 , 28 ].…”

RNA sequence analysis of dermal papilla cells’ regeneration in 3D culture

“…Besides, the features of 3D culture cells are closer to the complex in vivo condition 6 . Some researchers found that 3D cell cultures could induce pluripotent stem cells (iPSC) to generate personalized tissue models 7 . As the dermal papilla cells (DPCs) are unique mesenchymal cells, we previously found out that DPCs could also induce human hair growth in mice by transplanting intact DPCs and epithelial cells 4,8 .…”