The curious case of how mimicking physiological complexity in in vitro models of the human respiratory system influences the inflammatory responses. A preliminary study focused on gold nanoparticles

Abstract

Environmental and biomedical nanoparticles can pose potential health risks to the human respiratory system by inducing severe lung inflammation. The aim of this case study is to present a comparison of the inflammatory response in four in vitro models of the human lung epithelium, differing by composition and/or culturing substrates, when exposed to gold nanoparticles (AuNPs). Three in vitro models of lung adenocarcinoma (A549) cells and a commercially available three-dimensional (3D) culture (MucilAir ™ ) were tested.The models were exposed to AuNPs for 3, 6, and 24 h. AuNPs internalisation was investigated by confocal, electron microscopy, and Raman spectroscopy. Enzyme-Linked Immuno-Sorbent Assay (ELISA) was used for quantifying the secretion of the inflammatory mediator Interleukin-6 (IL-6) following exposure to AuNPs. Finally, a microfluidic approach was developed in-house to investigate whether pro-inflammatory mediators present in supernatants harvested from the AuNPs-treated cell cultures could trigger monocyte activation. Our results demonstrated that AuNPs were internalised only in submerged cultures grown on glass substrates. Nevertheless, AuNPs internalisation did not trigger a significant IL-6 secretion. Significant amounts of IL-6 were secreted by AuNPs-treated mono-cultures grown on Transwell ™ inserts, triggering monocyte activation in dynamic microfluidic experiments. AuNPs did not induce IL-6 secretion in co-cultures and MucilAir ™

Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials

“…This result suggests that there was a polarized secretion of cytokines by the epithelial cells into the apical compartment. This has been previously reported by other research groups for various human lung cell lines (including A549 cells) grown on Transwell™ membranes [ 26 , 49 , 50 , 51 ].…”

“…Cultures of human lung (A549) cells grown at the air–liquid interface (ALI) were tested as in vitro test model. A549 cells are an accepted model for the risk assessment of inhaled NP because they mimic the alveolar epithelium, which is the primary region where most inhaled NPs deposit [ 26 ]. Currently, in vitro models of the human lung epithelium used for NP risk assessment can vary in their complexity level [ 26 ].…”

“…A549 cells are an accepted model for the risk assessment of inhaled NP because they mimic the alveolar epithelium, which is the primary region where most inhaled NPs deposit [ 26 ]. Currently, in vitro models of the human lung epithelium used for NP risk assessment can vary in their complexity level [ 26 ]. In this study, A549 cells were tested as mono-cultures, in an attempt to narrow down the parameters influencing the cellular responses detected and highlight the influence of the administration mode in the nanomaterials risk assessment [ 26 ].…”

“…Currently, in vitro models of the human lung epithelium used for NP risk assessment can vary in their complexity level [ 26 ]. In this study, A549 cells were tested as mono-cultures, in an attempt to narrow down the parameters influencing the cellular responses detected and highlight the influence of the administration mode in the nanomaterials risk assessment [ 26 ]. When considering the human lung, in vitro models cultured in ALI conditions represent the more realistic screening tool currently available, considering the development and promotion of alternatives to animal experiments according to the “3Rs concept” of Russell and Burch: Replace, Reduce, and Refine [ 27 ].…”

Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs

“…Consistent with the scientific literature 42 – 46 , our results confirmed that the 3D spatial architecture of the cultures influenced the F-actin organization within the cells. While A549 cells in 2D are well-known to be characterized by F-actin stress-fibers 47 , in ALI multilayered mono-cultures the adenocarcinoma cell line showed a cortical organization of the F-actin (Fig. 2A , and Supporting video VS1).…”