The development and characterization of in vivo-like three-dimensional models of bronchial epithelial cell lines

Sara Van den Bossche; Lisa Ostyn; Valerie Vandendriessche; Charlotte Rigauts; Herlinde De Keersmaecker; Cheryl A Nickerson; Aurélie Crabbé

doi:10.1016/j.ejps.2023.106567

The development and characterization of in vivo-like three-dimensional models of bronchial epithelial cell lines

Eur J Pharm Sci. 2023 Nov 1:190:106567. doi: 10.1016/j.ejps.2023.106567. Epub 2023 Aug 24.

Authors

Sara Van den Bossche¹, Lisa Ostyn¹, Valerie Vandendriessche¹, Charlotte Rigauts¹, Herlinde De Keersmaecker², Cheryl A Nickerson³, Aurélie Crabbé⁴

Affiliations

¹ Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium.
² Centre of Advanced Light Microscopy, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium; Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium.
³ School of Life Sciences, Biodesign Center for Fundamental and Applied Microbiomics, Biodesign Institute, Arizona State University, 727 E. Tyler Street, Tempe, Arizona 85281, USA.
⁴ Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium. Electronic address: aurelie.crabbe@UGent.be.

PMID: 37633341
DOI: 10.1016/j.ejps.2023.106567

Abstract

In vitro models of differentiated respiratory epithelium that allow high-throughput screening are an important tool to explore new therapeutics for chronic respiratory diseases. In the present study, we developed in vivo-like three-dimensional (3-D) models of bronchial epithelial cell lines that are commonly used to study chronic lung disease (16HBE14o^-, CFBE41o^- and CFBE41o^- 6.2 WT-CFTR). To this end, cells were cultured on porous microcarrier beads in the rotating wall vessel (RWV) bioreactor, an optimized suspension culture method that allows higher throughput experimentation than other physiologically relevant models. Cell differentiation was compared to conventional two-dimensional (2-D) monolayer cultures and to the current gold standard in the respiratory field, i.e. air-liquid interface (ALI) cultures. Cellular differentiation was assessed in the three model systems by evaluating the expression and localization of markers that reflect the formation of tight junctions (zonula occludens 1), cell polarity (intercellular adhesion molecule 1 at the apical side and collagen IV expression at the basal cell side), multicellular complexity (acetylated α-tubulin for ciliated cells, CC10 for club cells, keratin-5 for basal cells) and mucus production (MUC5AC) through immunostaining and confocal laser scanning microscopy. Results were validated using Western Blot analysis. We found that tight junctions were expressed in 2-D monolayers, ALI cultures and 3-D models for all three cell lines. All tested bronchial epithelial cell lines showed polarization in ALI and 3-D cultures, but not in 2-D monolayers. Mucus secreting goblet-like cells were present in ALI and 3-D cultures of CFBE41o^- and CFBE41o^- 6.2 WT-CFTR cells, but not in 16HBE14o^- cells. For all cell lines, there were no ciliated cells, basal cells, or club cells found in any of the model systems. In conclusion, we developed RWV-derived 3-D models of commonly used bronchial epithelial cell lines and showed that these models are a valuable alternative to ALI cultures, as they recapitulate similar key aspects of the in vivo parental tissue.

Keywords: Bronchial epithelial cells; Cystic fibrosis; Differentiation; Organotypic; Rotating wall vessel bioreactor; Three-dimensional cell culture models.