Aline Chary

Commercial products incorporating graphene‐based two‐dimensional (2D) nanosheets are increasingly developed to enhance mechanical, thermal, and electrical performance. Potential release of inhalable particles during manufacturing or end‐of‐life processing, however, raises concerns regarding respiratory health. This study explored the pulmonary cytotoxicity and immunomodulatory activity of airborne particles generated by mechanical abrasion of industrially relevant few‐layer graphene (FLG) reinforced epoxy composites. A multimodal analytical strategy combining high‐resolution transmission electron microscopy, Raman microscopy, and time‐of‐flight secondary ion mass spectrometry enabled reliable detection and spatial mapping of FLG in pristine and abraded composites. Abrasion predominantly produced composite fragments in which FLG remained embedded, with no indication of protruding or freely released FLG nanosheets. Across advanced bronchial and alveolar epithelial air–liquid interface models, neither abraded epoxy nor FLG–epoxy particles induced cytotoxic or pro‐inflammatory responses. A moderate reduction of mucociliary clearance was observed only at high exposure doses of abraded epoxy particles, without amplification by FLG reinforcement. As‐produced FLG triggered strong TNF‐α secretion in alveolar macrophages, whereas abraded FLG–epoxy particles elicited minimal responses. Overall, the findings indicate limited acute respiratory toxicity associated with inhalation of fragments released from FLG‐based epoxy composites under production and end‐of‐life scenarios.

Single-cell mass cytometry in a 3D alveolar tetra-culture model uncovers cell-type and cluster-specific heterogeneity in silver nanoparticle association and downstream signaling, revealing mechanistic insights into NP cytotoxicity.

In recent years, a global increase in allergy incidence following chemical exposure has been observed. While the process of skin sensitization is well characterized through the adverse outcome pathway (AOP) framework, the immunological mechanisms underlying respiratory sensitization remain less well understood. Respiratory sensitizers are classified as substances of very high concern (SVHC) under the European Union (EU) regulation for the registration, evaluation, authorization and restriction of chemicals (REACH), emphasizing the importance of evaluating respiratory tract sensitization as a critical hazard. However, the existing new approach methodologies (NAMs) for the identification of skin sensitizers lack the capacity to differentiate between skin and respiratory sensitizers. Thus, it is imperative to develop physiologically relevant test systems specifically tailored to assess respiratory sensitizers. This study aimed to evaluate the efficacy of ALIsens^®, a three-dimensional (3D) in vitro alveolar model designed for the identification of respiratory sensitizers and to determine its ability to correctly identify sensitizers. In this study, we used a range of skin sensitizers and non-sensitizers to define the optimal exposure dose, identify biomarkers, and establish tentative thresholds for correct sensitizer classification. The results demonstrate that ALIsens^® is a promising in vitro complex model that could successfully discriminate respiratory sensitizers from skin sensitizers and non-sensitizers. Furthermore, the thymic stromal lymphopoietin receptor (TSLPr) cell surface marker was confirmed as a reliable biomarker for predicting respiratory sensitization hazards.

The human lung adenocarcinoma cell line A549 is commonly used in cancer research as a model of malignant alveolar type II epithelial cells. A549 cells are frequently cultured in Ham’s F12K (Kaighn’s) or Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with glutamine and 10% fetal bovine serum (FBS). However, the use of FBS presents significant scientific concerns, such as the presence of undefined components and batch-to-batch variation leading to possible reproducibility issues in experiments and readouts. This chapter describes how to transition A549 cells to FBS-free medium and gives some insights on the further characterizations and functionality assays that would be necessary to perform for the validation of the cultured cells. Key words Fetal bovine serum Cell culture Serum-free media Chemically defined media Xeno-free media Cell lines A549 Air–liquid interface

The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 μm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 μg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated. Significant cytotoxic effect was observed for all three types of AgNMs at the highest tested doses. The increased mRNA levels of the pro-apoptotic gene CASP7 suggests that apoptosis may occur after exposure to AgNWs. All three types of AgNMs increased the mRNA level of the anti-oxidant enzyme HMOX-1 and of the metal-binding anti-oxidant metallothioneins (MTs), with AgNWs being the most potent inducer. Even though all types of AgNMs induced the nuclear translocation of NF-kB, only AgNWs increased the mRNA level of pro-inflammatory mediators. The pro-inflammatory response elicited by AgNWs was further confirmed by the increased secretion of the 10 evaluated interleukins. In the current study, we demonstrated that the direct exposure of a complex tetra-culture alveolar model to different types of AgNMs at ALI induces shape- and size-specific biological responses. From the three AgNMs tested, AgNWs were the most potent in inducing biological alterations. Starting from 50 ng/cm2, a dose representative for an acute exposure in a high exposure occupational setting, AgNWs induced prominent changes indicative for a pro-inflammatory response. Even though the acute responses towards a dose representative for a full-lifetime exposure were also evaluated, chronic exposure scenarios at low dose are still unquestionably needed to reveal the human health impact of AgNMs during realistic conditions.

During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells. Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture. In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies.