Aerosol–Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction

April 15, 2022

Mélanie M. Leroux 1 , Romain Hocquel 1, Kevin Bourge 1 , Boštjan Kokot 2 , Hana Kokot 2 , Tilen Kokliˇc 2, Janez Štrancar 2, Yaobo Ding 3 , Pramod Kumar 3, Otmar Schmid 3 , Bertrand H. Rihn 1, Luc Ferrari 1 and Olivier Joubert 1
1 Institut Jean Lamour, UMR CNRS 7198, Université de Lorraine, CNRS, IJL, F-54000 Nancy, France
2 Jožef Stefan Institute, Department of Condensed Matter Physics, 1000 Ljubljana, Slovenia
3 Institute of Lung Health and Immunity, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany

The goal of this work was to refine an ALI pre-established protocol for the VITROCELL® Cloud 6 system. Therefore, after addressing each crucial step, (i.e., the use of very low concentrations of NP, the deposition of a homogeneous layer of surfactant, the specific use of semi-adherent cells, and finally the enhancement of the extraction yield of RNA to carry out transcriptomic studies), we present here for the first time a robust almost ready-to-use protocol. This one will be helpful for nanotoxicologists interested in developing such methodologies.


Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air–liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. Objectives: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. Methods: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. Results: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 µL of 30 mg/mL TiO2 and 114 µL of 15 mg/mL TiO2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 µL.

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