Poster: Human bronchial organotypic culture

Response of human bronchial organotypic culture to mainstream cigarette smoke exposure

R. Kostadinova, C. Mathis, S. Frentzel, D. Kuehn, S. Majeed, C. Merg, A. Elamin, E. Guedj, R. Dulize, Y. Xiang, P. Leroy, M.C. Peitsch, J. Hoeng / Philip Morris International Research and Development, Philip Morris, Switzerland


R. Kostadinova, Philip Morris International Research and Development

OpenTox Euro 2014 Poster: Human bronchial organotypic culture

The goal of our study is to combine a state-of-the art systems toxicology approaches with a relevant human in vitro airway model which mimics closely the in vivo situation (C.Mathis et al., 2013) to investigate the impact of direct cigarette smoke (CS) exposure on respiratory tract epithelium tissues. Additionally we aim to determine the robustness of our study design by analysing multiple end points of two batches of airway tissue cultures exposed in the same way to separate mainstream CS exposures. We used differentiated human bronchial epithelial tissue model (EpiAirwayTM, MatTek®) produced by culturing human primary cells at the air-liquid interface. This tissue culture consist of basal cells, mucus secreting cells and ciliated cells and show an in vivo-like structure.

Bronchial organotypic epithelial tissue culture was exposed for 28 min at the air-liquid interface to fresh air or to 8%/15% (vol/vol with air) diluted mainstream CS (reference cigarette 3R4F; Health Canada smoking regimen and nicotine doses of 0.15mg/L/0.25mg/L). Different endpoints (adenylate kinase (AK) assay, cytochrome P450 CYP1A1/CYP1B1 activity, histology/immunostaining, pro-inflammatory markers release and gene expression) were then collected at 4h, 24h, 48h and 72h after exposure to assess time- and dose-dependent effect of whole CS exposure.

Our results demonstrated that 15% of CS induced cytotoxicity in the bronchial tissues measured by the release of AK in the media at 24h, 48h and 72h post-exposure times. Histological and immunohistochemical analysis showed that 3R4F induced dose- and time- dependent bronchial tissue damage characterized with reduction of the tissue thickness and decrease of the number of proliferating and ciliated cells.

Furthermore CS induced time- and dose-dependent increase of the CYP1A1/1B1 activities (at 48h and 72h post-exposure time points) and of the release of pro-inflammatory mediators such as VEGF, MMP-1, MMP-9, GM-CSF, IL-1α, IL-6, IL-8, IP-10, sICAM-1, TNF-α and GRO at 24h, 48h and 72h post-exposure time points). Transcriptomic analyses combined with quantitative biological network modeling indentified biological processes (e.g. inflammation, proliferation and cellular stress) which were triggered by acute CS exposure in the bronchial tissue culture at various post-exposure time points.

Finally, good reproducibility of the obtained results (AK assay, CYP1A1/CYP1B1 activity, pro-inflammatory markers release and gene expression) was observed in two independent experiments.

The described study and the reproducible data obtained from two independent experiments suggest that combining biological end points, large scale molecular measurements and computational method is a powerful approach to assess CS effect using a relevant in vitro model such as bronchial epithelial tissue culture exposed at the air-liquid interface.