Development of Apical-out Airway Organoids to Evaluate Respiratory Toxicity of Polystyrene Microplastics

Background: Even though it has been reported that microplastic could infiltrate in the lung and induce inflammation, exact pathophysiology of microplastic indued-lung injuries has not been fully revealed.One of the main hurdles for evaluating respiratory exposure effect of microplastic on the airway is shortage of in vitro models which are relevant with human exposure of microplastic during inhalation.In this study, we generated apical out airway organoid (AOAO) which epithelial cell located in outer surface of organoid to evaluate the feasibility of AOAO for respiratory exposure models of microplastics.Methods: Human airway basal stem cells (hABSCs, passage 2) were trypsinized and resuspended (10,000 cells/mL) in Pneuma Cult-ALI Medium (#05001; STEMCELL Technologies, Canada) supplement with Pneuma Cult-ALI supplements, hydrocortisone, and heparin.One hundred L of resuspended hABSCs were placed per well in a low attachment 96-well microplate (Corning #7007, USA) and centrifuged at 1,500 RPM for 15 minutes.Fifty L of medium were removed and refreshed every 2 days.The cultures were maintained at 37 C with 5% CO2 for 21 days, and generation and movement of AOAOs were video recorded at 7-day intervals using an EVOS M7000 Imaging System (ThermoFisher Scientific, USA).After generating AOAO, polystyrene (PS) was treated and inflammation response were evaluated.Results: To characterize AOAO in the resulting day 21, we performed immunofluorescence staining of key markers of airway epithelium and observed highly selective localization of acetylated-alpha-tubulin on the organoid outer surface.Besides ciliated cells, other cell types of airway epithelium such as basal cells and goblet cells were identified.Moreover, goblet cells and basal cells were localized on the inner layer of AOAOs.We then calculated the length of cilia on the organoid's outer surface.Similar to the human airway tissue, AOAOs have beating cilia.By treating PS, goblet cell hyperplasia was shown in the AOAO.Expression of inflammatory cytokine such as tumor necrosis factor- and interleukin-1 were increased by PS.Conclusion: Apical-out airway organoids mimic human upper airway epithelium and cilia function.Moreover, AOAOs represent an increase of mucin secretion and inflammation following microplastic exposure.