A novel, easy-to-use cell exposure system (ALICE) for controlled pharmaco-toxicological studies with liquids at the air-liquid interface

Introduction: Typically, pharmacological and toxicological in vitro cell studies are performed under submerged cell conditions, i.e. the substance of interest is mixed directly into the cell culture medium. However for cell systems located at the air-liquid interface (e.g. lung epithelium, skin), controlled cell exposures at the air-liquid interface are more realistic.

Methods: Here we present a technically simple, yet dosimetrically accurate air-liquid interface cell exposure system (ALICE) for controlled thin-film deposition of liquids or colloidal nanoparticle onto cells. The ALICE consists of a vibrating membrane droplet generator (customized eFlow, Pari, Munich, Germany), an exposure unit and a conditioning system, which provides temperature and humidity conditions suitable for cell cultivation (to 37°C and 80–95% relative humidity).

Results: The system allows uniform and efficient deposition (57±7%) of nebulized solutions and nanoparticle suspensions onto the bottom of the exposure chamber, where up to two cell culture plates with transwell inserts can be placed. The deposition mechanism is a unique combination of cloud settling and single particle sedimentation. Up to 5ml of these substances can be nebulized and deposited within 10min forming a uniform, thin substance film (14µm thickness per 1ml sprayed liquid) on the cells at the air-liquid interface. Exposures can be repeated with a high degree of reproducibility in deposited particle/solute mass (variability <12% relative to mean deposited mass) and the transwell inserts can easily be removed and processed for measuring cell response parameters. Initial tests with cultured A549 lung epithelial cells have shown no significant impairment of cell viability during the exposure in the ALICE. On the other hand, significant differences in cellular mRNA/protein response (IL-8, HO-1) were observed when comparing air-liquid interface and submerged cell exposure with zinc oxide (ZnO) nanoparticles.

Discussion: This indicates that pharmaceutical and toxicological studies with inhaled nanoparticles should be performed under the more realistic air-liquid interface rather than submerged cell conditions.