106 Establishing a Physiologically Relevant in Vitro Airway Model to Assess the Impacts of Environmental Exposures, Like Smoking, on Covid-19 Susceptibility.

Abstract

Abstract Air-liquid interface (ALI) cultures are commonly used for 3D modelling of the human airways and to examine the effects of direct exposure to airborne toxicants. We explored the suitability of human epithelial cell lines (RPMI 2560, HBEC-3KT, CALU-3 and A549) and primary cells (nasal, bronchiolar and small airways) grown at ALI as a physiologically relevant model to examine whether environmental exposures can increase the susceptibility of the human airways to SARS-COV-2 infection. Cultures were kept at ALI for up to 28 days to allow differentiation. Trans-epithelial electrical resistance (TEER) was measured weekly. Cells were harvested at 0, 7, 14, 21 and 28 days, and transcriptional markers of basal, ciliated and mucus-producing cells together with SARS-COV-2 viral entry genes (ACE2, TMPRSS2 and Furin) and other genes of interest, were assessed by reverse-transcription quantitative PCR. Each cell line yielded differentiated models, with upregulation of markers of ciliated and mucus producing cells from day 7. In contrast to primary cells, immortalised cell lines did not produce cilia but, mucus was produced. TEER values increased with time, as expected, but values and therefore formation of tight junctions differed between models. Similarly, expression of viral entry genes increased as cells differentiated with marked differences across the different models. This study will be used to select the most appropriate ALI model for investigating the effect of environmental exposures such as smoking and vaping on susceptibility to SARS-COV-2 infection.