Methodological approaches to test complex mixtures in vitro

Application of in vitro methods for analyzing the toxicological effects of gases and complex mixtures is still limited due to the fact that there are no generally accepted concepts and techniques for effective exposure of adherent growing cells to test atmospheres. This is especially true for gaseous and particulate materials. Therefore, we established a novel experimental approach using an exposure device based on the cell cultivation system CULTEX. This allowed us to investigate chemically and physically unchanged atmospheres in an in vitro system by exposing target cells cultured directly at an air/liquid interface. The exposure device is flexible enough to be connected to different aerosol-generating systems independent of an incubator. During exposure, all requirements for maintaining cell viability are fullfilled. In order to demonstrate the applicability and sensitivity of this in vitro approach in detecting dose-dependent effects of airborne substances, human lung epithelial cells were exposed to either clean air or air contaminated with single gases (ozone, nitrogen dioxide) or complex mixtures (e. g. sidestream and mainstream cigarette smoke). Cellular reactions dependent on dose or dilution were established in a reproducible manner in all cases; this also demonstrated the stability of the system. The biological parameters estimated were mainly cell number, metabolic activity and glutathione content. The introduction of this experimental system in the field of inhalation toxicology offers new test strategies for the in vitro evaluation of a wide range of inhalable substances in both laboratory and ambient situations