In vitro assessment of ferroptosis of cells exposed to cigarette smoke aerosol using a self-designed on-chip evaluation system based on gas-liquid dual-dimensional exposure.

Aerosol pollutants significantly cause health concerns. Herein, we established an original real-time aerosol exposure system that used a self-designed bionic-lung microfluidic chip. The chip features a 4 × 4 intersecting array within gas and liquid layers, creating 16 distinct microenvironments. A membrane situated between the layers offers attachment for cells and establishes a gas-liquid interface. This design provides a reliable screening capacity for investigating the biological effects of aerosol exposure in vitro by manipulating the gas and/or liquid conditions. Using this system, we validated that cigarette smoke (CS) aerosol triggered a concentration- and time-dependent reduction in cell viability and intracellular glutathione levels, accompanied by an increase in intracellular reactive oxygen species and Fe2+. Furthermore, CS aerosol significantly downregulated the expression of GPX4, SLC7A11, and FTL mRNA while inducing a notable increase in that of ACSL4 mRNA. Additionally, CS aerosol markedly stimulated the release of proinflammatory cytokines. Crucially, the ferroptosis inhibitor deferoxamine mesylate reversed these biological indicators. These results demonstrate that our novel bionic-lung chip presents a suitably achievable approach to investigate the biological effects induced by aerosol exposure.

Investigation of the in vitro toxic effects induced by real-time aerosol of electronic cigarette solvents using microfluidic chips.

The safety of propylene glycol (PG) and vegetable glycerin (VG) as solvents in electronic cigarette liquid has received increasing attention and discussion. However, the conclusions derived from toxicity assessments conducted through animal experiments and traditional in vitro methodologies have consistently been contentious. This study constructed an original real-time aerosol exposure system, centered around a self-designed microfluidic bionic-lung chip, to assess the biological effects following exposure to aerosols from different solvents (PG, PG/VG mixture alone and PG/VG mixture in combination with nicotine) on BEAS-2B cells. The study aimed to investigate the impact of aerosols from different solvents on gene expression profiles, intracellular biomarkers (i.e., reactive oxygen species content, nitric oxide content, and caspase-3/7 activity), and extracellular biomarkers (i.e., IL-6, IL-8, TNF-α, and malondialdehyde) of BEAS-2B cells on-chip. Transcriptome analyses suggest that ribosomal function could serve as a potential target for the impact of aerosols derived from various solvents on the biological responses of BEAS-2B cells on-chip. And the results showed that aerosols of PG/VG mixtures had significantly less effect on intracellular and extracellular biomarkers in BEAS-2B cells than aerosols of PG, whereas increasing nicotine levels might elevate these effects of aerosol from PG/VG mixture.

Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Characterisation of a smoke/ aerosol exposure in vitro system (SAEIVS) for delivery of complex mixtures directly to cells at the air-liquid interface.

In vitro testing is important to characterise biological effects of consumer products, including nicotine delivery products such as cigarettes, e-cigarettes and heated tobacco products. Users' cells are exposed to these products' aerosols, of variant chemical compositions, as they move along the respiratory tract. In vitro exposure systems are available to model such exposures, including delivery of whole aerosols to cells, and at the air-liquid interface. Whilst there are clear advantages of such systems, factors including time to aerosol delivery, aerosol losses and number of cell cultures that can be exposed at one time could be improved. This study aimed to characterise a custom-built smoke/ aerosol exposure in vitro system (SAEIVS) using 1R6F reference cigarette smoke. This system contains five parallel smoking chambers and delivers different dilutions of smoke/ aerosol to two separate cell culture exposure chambers in <10 s. Using two dosimetry measures (optical density 400 nm [OD400 ]; mass spectrometric nicotine quantification), the SAEIVS demonstrated excellent linearity of smoke dilution prior to exposure (R2  = 0.9951 for mass spectrometric quantification; R2  = 0.9965 for OD400 ) and consistent puff-wise exposures across 24 and 96 well plates in cell culture relevant formats (e.g., within inserts). Smoke loss was lower than previously reported for other systems (OD400 : 16%; nicotine measurement: 20%). There was good correlation of OD400 and nicotine measurements, indicating that OD was a useful surrogate for exposure dosimetry for the product tested. The findings demonstrated that the SAEIVS is a fit-for-purpose exposure system for the reproducible dose-wise exposure assessment of nicotine delivery product aerosols.

New application for assessment of dry eye syndrome induced by particulate matter exposure.

Dry eye syndrome (DES) is a multifactorial condition characterized by insufficient tear lubrication and eye irritation. Air pollutants, including particulate matter (PM), are an emerging threat to human health causing DES and other diseases. However, the pathogenic mechanisms of DES induced by PM exposure remain to be fully elucidated. Recent studies have attempted to create DES animal model using PM exposure. In this study, we explored a novel in vivo exposure model of DES, utilizing an inhalation device (aerosol exposure system) to reproduce the natural exposure to atmospheric PM. Rats were exposed to urban PM (UPM) using this aerosol system for 5 h per day over 5 days. Tear volume in UPM-exposed rats decreased significantly, whereas corneal irregularity and lissamine green staining significantly increased following UPM exposure. Additional effects observed following UPM exposure included apoptosis in the corneal epithelium and a decrease in the number of goblet cells in the conjunctiva. UPM also affected the stability of the tear film by disrupting its mucin-4 layer. In conclusion, aerosol exposure systems have proven effective as assessment tools for DES caused by PM.

Development and testing of a new-generation aerosol exposure system: The independent holistic air-liquid exposure system (InHALES).

The dose of inhaled materials delivered to the respiratory tract is to a large extent a function of the kinetics of particle deposition and gas dissolution on or in the airway and lung epithelia, and therefore of the structural and functional properties of the respiratory tract. In vitro aerosol exposure systems commonly do not simulate these properties, which may result in the delivery of non-realistic, non-human-relevant doses of inhalable test substances to the in vitro biological test systems. We developed a new-generation in vitro aerosol exposure system, the InHALES, that can, like the human respiratory tract, actively breathe, operate medical inhalers, or take puffs from tobacco products. Due to its structural and functional similarity to the human respiratory tract, the system is expected to deliver human-relevant doses of inhalable materials to cell cultures representing respiratory tract epithelia. We here describe the proof of concept of the InHALES with respect to aerosol delivery and compatibility with oral, bronchial, and alveolar cell cultures. The results indicate that the system structure and function translate into complex patterns of test atmosphere delivery that, with increasing system complexity, may closely mimic the patterns observable in the human respiratory tract.

Characterization of the Vitrocell® 24/48 aerosol exposure system for its use in exposures to liquid aerosols.

BACKGROUND: The Vitrocell® 24/48 is an advanced aerosol exposure system that has been widely used and characterized for exposure studies of cigarette smoke, but not for exposure to liquid aerosols with a low gas-vapor phase content such as the ones generated by electronic cigarettes. An experimental system characterization for this specific application was therefore performed. METHODS: Glycerol model aerosols of different particle size distributions, produced by a condensation monodisperse aerosol generator, were used for exposing small volumes of phosphate-buffered saline in the Vitrocell® 24/48. Disodium fluorescein, added as a tracer in the aerosol, allowed the exact aerosol mass deposition to be quantified fluorometrically. RESULTS: The aerosol mass delivery efficiency within the system showed variations in the range of ±25%. Aerosol dilution was not fully reflected in aerosol delivery, the achieved aerosol delivery should therefore be determined experimentally. Quartz crystal microbalances underestimated the deposition of liquid aerosols. Unequal delivery of particles of different sizes was detectable, although this effect is unlikely to be relevant under applied experimental conditions. CONCLUSIONS: The Vitrocell® 24/48 aerosol exposure system can be used for exposures to liquid aerosols, such as those generated by electronic cigarettes. However, our results indicate that, compared with aerosol studies of cigarettes, a higher variability is to be expected.

A new fluorescence-based method for characterizing in vitro aerosol exposure systems.

Knowledge of how an in vitro aerosol exposure system delivers a test aerosols to the biological test system is among the most crucial prerequisites for the interpretation of exposure experiments and relies on detailed exposure system characterization. Although various methods for this purpose exist, many of them are time consuming, require extensive instrumentation, or offer only limited ability to assess the performance of the system under experimental settings. We present the development and evaluation of a new, highly robust and sensitive fluorometry-based method for assessing the particle size specific delivery of liquid aerosols. Glycerol aerosols of different mean particle sizes and narrow size distributions, carrying the fluorophore disodium fluorescein, were generated in a condensation monodisperse aerosol generator. Their detailed characterization confirmed their stability and the robustness and reproducibility of their generation. Test exposures under relevant experimental settings in the Vitrocell® 24/48 aerosol exposure system further confirmed their feasibility for simulating exposures and the high sensitivity of the method. Potential applications of the presented method range from the experimental confirmation of computationally simulated particle dynamics, over the characterization of in vitro aerosol exposure systems, to the detailed description of aerosol delivery in test systems of high complexity.