In vitro exposure of isolated cells to native gaseous compounds Development and validation of an optimized system for human lung cells

Abstract

An exposure system for adherent growing cells to native gaseous compounds was developed using air/liquid culture techniques on the basis of the Cultex system'. In contrast to other exposure systems the reproducible testing of native environmentally relevant gases without changing their physical or chemical properties including heating, CO2- content and humidity is possible. Specially designed systems for medium flow and gas support guarantee the nutrification and humidification as well as the direct gas contact of the exposed cells which are cultivated on microporous membranes (0.4 microm pore size). The system works independently of a cell culture incubator offering the possibility to analyze any relevant gas mixture directly under indoor or outdoor conditions. Several experimental approaches were carried out to characterize the properties of the system. In exploratory experiments without cells, the reproducibility and quality of the gas/membrane contact could be demonstrated. Exposures of human lung fibroblasts (Lk004 cells) and human lung epithelial cells (HFBE-21 cells) to synthetic air, ozone (202 ppb, 510 ppb) and nitrogen dioxide (75 ppb to 1,200 ppb) established that cells could be treated for 120 minutes without significant loss of cellular viability. At the same time, the experiments confirmed that such exposure times are long enough to detect biological effects of environmentally relevant gas mixtures. The analysis of viability (viable cell number, tetrazoliumsalt cleavage) and intracellular end-points (oxidized/reduced glutathione, ATP/ADP) showed that both gases induced relevant cellular changes. In summary, the efficiency and practicability of this newly developed exposure system for adherent human lung cells could be clearly demonstrated.