Impact of liquid dosing and associated cell ‘drowning’ in a 3D human bronchial epithelial model used for protease exposure studies

Introduction As part of a programme of work developing human-relevant New Approach Methodologies (NAMs) for next-generation risk assessment (NGRA), use of a human air-liquid interface (ALI) bronchial epithelial model (MucilAir™) to investigate the impact of exposure to protease was assessed. Methods To guide in vitro dosing, scenarios representing low-high risk of sensitisation after 8 h of protease exposure in an occupational setting were modelled by performing simulations with the Multiple Path Particle Dosimetry (MPPD) model with refinement of the outputs using a tracheobronchial/alveolar clearance model, to estimate tracheobronchial tissue doses. With no effects seen at these concentrations, higher concentrations were subsequently tested to identify thresholds for biological effects. Repeated apical liquid exposures over 8 h were applied using a wide concentration range of protease (0.00125–7812 μg protease protein/mL) or phosphate-buffered saline (PBS) control. Effects on epithelial barrier integrity, cytokine production, and extracellular vesicle (EV) dynamics were measured. Results Reduced transepithelial electrical resistance (TEER), increased EV and IL-8 secretion were observed using a PBS control reflecting the impact of liquid dosing alone. No significant protease-related adverse effects were observed at concentrations of 0.5 μg protease protein/mL or lower when compared to the PBS control. At concentrations of 75 μg protease protein/mL or more, however, TEER was significantly reduced and mucin and tetraspanin expression on EVs was degraded. Discussion Here, we show the impact of liquid dosing when investigating the effects of materials on bronchial epithelia, and challenges encountered when working with proteases. This work provides a foundation for developing in vitro methodology for generation of data for use in risk assessment of inhalation of enzymes and other materials. It is hypothesised that nebulised protease delivery could be a more suitable alternative and better replicate in vivo (human) inhalation dynamics.