Biological effects of inhalable compounds improvements of the in vitro testing method

The state-of-the-art technique for testing inhalable gases in vitro is the air-liquid-interphase (ALI) method. The concept is based on the cultivation of cells on microporous membranes and can result in an efficient contact to airborne test atmospheres under controlled conditions. Various biological models including cell lines, primary cells, co-cultures and complex ex-vivo models can be applied. Round-robin prevalidation studies with model gases demonstrated the sensitivity and relevance of the basic technique in principle. However, until now major limiting factors still exist including robustness, practicability, applicability and efficacy of the method regarding aerosol applications. A limited control of the individual cell exposure and a lack of more complex routine cell models also still prevent a general acceptance of this promising method for routine testing. In a first step we therefore focused on the improvement of the technique concerning (i) the integration of the complex exposure process in a smooth lab workflow, (ii) the incorporation of a cell culture model with an artificial lung surfactant and (iii) the deposition of airborne particulate matter for aerosol testing. As a result, an exposure device and procedure was developed permitting a more uniform and smooth processing of cells than the common technique. By that way, the handling of the biological test systems can be conducted in a more appropriate way leading to a higher robustness, practicability and ease of use. Nowadays, the presence of surfactant as a part of the biological model is generally accepted as essential for the relevance of the in vitro testing in inhalation toxicology. To realize applicability for routine testing at the same time, a cell culture model, including A549 cells and an artificial fluid composed comparably to natural lung surfactant was applied. Results of a first series of exposures using test gases show that this model can be used in such an exposure regime. Moreover, typical protective characteristics of the surfactant could be documented. To increase the applicability of the ALI-method in aerosol testing, numerical simulations (CFD) were applied for the optimization of the particle transportation. In the physical models of the simulation a combination of different physical deposition mechanisms was regarded. First results of simulation and laboratory experiments showed a clearly increased deposition efficiency of particles from aerosols in the test system. At the same time, no adverse effects of the deposition procedure were observed in the exposed cells. Therefore, the improvement of the procedure so far seems promising to further enhance the applicability of alternative methods in the study of inhaled substances significantly.