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

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™ models, although supernatants harvested from co-cultures triggered monocyte activation. Our case study demonstrates that in vitro complexity, as well as culturing substrates, deeply influence the detectable cellular responses to nanoparticles, and advocate for the adoption of more advanced tissue-mimetic cultures of the human respiratory system for nanomaterials testing.