Robust, efficient workflow to establish, culture, and functionally assess primary-isolated airway epithelial cells

ABSTRACT

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s) Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s) Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s) In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved