Journal of Visualized Experiments www.jove.com Copyright © 2020 Journal of Visualized Experiments February 2020 | 156 | e60572 | Page 1 of 12 Video Article Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface Amelie Tsoutsoulopoulos 1 , Katrin Gohlsch 2 , Niklas Möhle 3 , Andreas Breit 2 , Sebastian Hoffmann 5 , Olaf Krischenowski 3,4 , Harald Mückter 2 , Thomas Gudermann 2 , Horst Thiermann 1 , Michaela Aufderheide 3,4 , Dirk Steinritz 1,2 1 Bundeswehr Institute of Pharmacology and Toxicology 2 Walther Straub Institute of Pharmacology and Toxicology, University of Munich 3 Cultex Laboratories GmbH 4 Cultex Technologies GmbH (formerly Cultex Laboratories GmbH) 5 seh consulting + services Correspondence to: Amelie Tsoutsoulopoulos at amelietsoutsoulopoulos@gmail.com URL: https://www.jove.com/video/60572 DOI: doi:10.3791/60572 Keywords: Medicine, Issue 156, Acute pulmonary toxicity, in vitro, exposure system, air-liquid interface, validation, cytotoxicity, airborne particles Date Published: 2/23/2020 Citation: Tsoutsoulopoulos, A., Gohlsch, K., Möhle, N., Breit, A., Hoffmann, S., Krischenowski, O., Mückter, H., Gudermann, T., Thiermann, H., Aufderheide, M., Steinritz, D. Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air- Liquid Interface. J. Vis. Exp. (156), e60572, doi:10.3791/60572 (2020). Abstract Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment. Video Link The video component of this article can be found at https://www.jove.com/video/60572/ Introduction Inhalation of toxic airborne particles is a public health concern, leading to a multitude of health risks worldwide and many millions of deaths annually 1,2 . Climate change, the ongoing industrial development and the rising demand for energy, agricultural and consumer products have contributed to the increase of pulmonary diseases over the last years 3,4,5,6 . Knowledge and evaluation of inhalable substances regarding their acute inhalation toxicity provide the basis for hazard assessment and risk management, but this information is still lacking for a wide range of these substances 7,8 . Since 2006, the EU chemical legislation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) requires that already existing and newly introduced products undergo a toxicological characterization including the inhalation route before being placed on the market. Therefore, REACH focuses on alternative and animal-free methods, the implementation of the "3R" principle (Replacement, Refinement, and Reduction of animal experiments) and the use of appropriate in vitro models 9 . In recent years, many different and adequate non-animal inhalation toxicity testing models (e.g., in vitro cell cultures, lung-on-a-chip models, precision cut lung slices (PCLS)) have been developed in order to assess the acute inhalation toxicity of airborne particles 5,7,10,11 . In terms of in vitro cell culture models, cultivated cells can be exposed under submerged conditions or at the ALI (Figure 1). However, the validity of submerged exposure studies is limited with regard to the evaluation of the toxicity of airborne compounds especially particles. Submerged exposure techniques do not correspond to the human in vivo situation; the cell culture medium covering the cells may affect the physico-chemical properties and thus, the toxic properties of a test substance 12,13 . ALI in vitro inhalation models allow the direct exposure of cells to the test substances without interference of the cell culture medium with test particles, thus, mimicking human exposure with higher physiological and biological similarity than submerged exposures 12,14 . For regulatory processes such as REACH, however, only animal models are available in the field of acute inhalation toxicology, as no alternative in vitro methods have been sufficiently validated and officially accepted so far 14 . For this purpose, test models have to be validated according to the requirements of the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) principles on test validity 15 .