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

ABSTRACT: 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.

SUBMITTER: Leroux MM 

PROVIDER: S-EPMC9028274 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature