Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials

March 15, 2023

DOI: 10.1002/smll.202207207

Yvonne Kohl1, Michelle Müller1, Marielle Fink2, Marc Mamier2, Siegfried Fürtauer3, Roland Drexel4, Christine Herrmann5, Stephan Dähnhardt-Pfeiffer6, Ramona Hornberger3, Marius I. Arz3, Christoph Metzger5, Sylvia Wagner1, Sven Sängerlaub3, Heiko Briesen5, Florian Meier4, and Tobias Krebs2

1Bioprocessing & Bioanalytics Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280 Sulzbach, Germany
2VITROCELL Systems GmbH, Fabrik Sonntag 3, 79183 Waldkirch, Germany
3Materials Development Fraunhofer Institute for Process Engineering & Packaging IVV, Giggenhauser Str. 35, 85354 Freising, Germany
4Postnova Analytics GmbH, 86899 Landsberg am Lech, Germany
5Process Systems Engineering School of Life Sciences Technical University Munich, Gregor-Mendel-Str. 4, 85354 Freising, Germany
6Microscopy Services Dähnhardt GmbH, Plambeckskamp 2, 24220 Flintbek, Germany

The goal of this study was to develop and characterize a 96-well aerosol exposure system for the safety assessment of nanomaterials. For this purpose, an exposure system for sequential nebulization in a 96-well format was designed, fabricated, and characterized using an ALI lung epithelial model. This 96-well exposure system was developed with the capability of sequential exposure of individual rows of wells with test substances as aerosols, thus offering the possibility of creating dose-response curves using the system. This allows the generation of a more reliable cell-based assay data for many types of applications, such as safety analysis, disease modeling, drug efficacy testing, or chemical registration.


In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air–liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air–liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cellbased assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivolike and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

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