title: Optimization of the CULTEX(®) radial flow system for in vitro investigation of lung damaging agents.
abstract: Exposure of the respiratory tract to airborne particles is gaining more and more importance due to the ubiquitous application of these particles in the field of industry, pharmacy and in daily life. Remarkably, the toxic properties and the underlying pathomechanisms with regard to inhalable substances have been insufficiently investigated so far. Thus, the EU Chemicals Regulation demands toxicological data (including the identification of potential inhalation hazards) for all chemicals placed on the market until 2018 (REACH). This requires extensive, technically complex and expensive inhalation toxicology studies that are usually generated in animal experiments. However, the legislation demands the consideration of the "3Rs" principle. Thus, in vitro-based test systems for the assessment of pulmonary toxicity are required. One promising approach to assess acute pulmonary toxicity of airborne particles is the CULTEX(®) RFS methodology that allows exposure of human lung epithelial cells at the air-liquid interface mimicking the alveolar situation. A prevalidation study showed the general applicability of this method. However, the clean air exposure group, which served as unexposed controls, exhibited some variations with regard to cell viability compared to the incubator control group. The aim of this study was therefore the identification of the possible causes and the improvement of methodological aspects. Several parameters including the general workflow, adjustment of airflow parameters, and cleaning procedures were investigated and adapted. Finally, our results showed the successful optimization of the CULTEX(®) RFS methodology for clean air exposure of A549 cells. However, although viability data in incubator controls and clean air exposures were equal, a distinct difference in cell morphology was observed that required further optimization. Additional experiments identified that open-wall cell culture inserts with a 2-fold pore density were found to be superior compared to the standard inserts and thus the deciding factor for the improvement of cell morphology. The presented findings are an important step in providing the CULTEX(®) RFS methodology as a promising alternative method to current in vivo testing in inhalation toxicology.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany. Electronic address: amelietsoutsoulopoulos@gmail.com.
authors.forename: Amelie
authors.initials: A
authors.lastname: Tsoutsoulopoulos
authors.affiliation: Cultex(®) Laboratories GmbH, Feodor-Lynen-Straße 21, 30625 Hannover, Germany.
authors.forename: Niklas
authors.initials: N
authors.lastname: Möhle
authors.affiliation: Cultex(®) Laboratories GmbH, Feodor-Lynen-Straße 21, 30625 Hannover, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Aufderheide
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, 80336 Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
mesh_terms: D000826:Animal Testing Alternatives; D015824:Blood-Air Barrier; D018929:Cell Culture Techniques / Q000295:instrumentation*; D045744:Cell Line, Tumor; D048430:Cell Shape; D002470:Cell Survival; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004867:Equipment Design; D006801:Humans; D006813:Humidity; D019570:Inhalation Exposure; D008567:Membranes, Artificial; D016062:Porosity; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D018675:Toxicity Tests / Q000295:instrumentation* / Q000379:methods
chemical_list: D008567:Membranes, Artificial
keywords: Air-liquid interface; CULTEX(®) RFS; Humidity; Lung in vitro models; Risk assessment; Toxicology