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( A ) Overview of the exhaust emission exposure system with an example vehicle (scooter) on the chassis dynamometer ( 1 ), the rotating disk diluter ( 2 ), instrumentation for the characterisation of the particulate compounds ( 3 ) and the exposure box ( 4 ). ( B ) The exposure box with the vacuum pump at the end of the system ( 5 ) and the heated water chamber for the humidification ( 6 ). ( C ) Exposure (right) and reference (left) chambers for the exposure of cell cultures and the condition control measurement units ( 7 ) in the interior of the exposure box. Image adapted from Muller et al . (38).
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In light of the increasingly abundant use of engineered nanoparticles (NPs) and the ongoing exposure to ambient ultrafine particles it is imperative that the potential for NPs to elicit adverse effects on human health is understood. In order to determine the potential harm that NPs may exert, many different in vitro systems have been used. Commonly...
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Citations
... Since the use of animals, mainly rodents, in toxicological practices poses ethical and social concerns (Russell and Burch, 1959) and is not aligned with the 3 R principles, the development of new approach methodologies in inhaled particulate matter health assessment is of paramount importance in term toxicant-target interactions, fate and biological response (Lazaridis, 2023). The use of complex in vitro lung models offers a relevant alternative to in vivo studies for predicting the acute toxicity of inhaled substances (such as gases, volatile organic compounds, PAH and particulate matter including nanoparticles) (Müller et al., 2011;de Souza Carvalho et al., 2014a,b;Jaber and Billet, 2023). However, their suitability for representing the different part of the respiratory system, which comprises around forty different cell types is not widely accepted. ...
... In recent years, nanoparticles and nanocolloids have acquired greater interest due to their extensive application in numerous clinical and business fields, including pharmaceuticals, biosensors, magnetic equipment, printing industries, and similar areas. Nanoparticles have attracted a lot of attention due to their high surface free energy, strong chemical activity, high reactivity due to their small particle size, very high specific surface area, and large adsorption capacity (Pokropivny et al., 2007;Müller et al., 2011). ...
Ion flotation is one of the most promising and unique methods for reducing or removing toxic heavy metal ions, organic pollutants, or inorganic anions and cations from mining and metallurgical wastewaters. It is a cost-effective and convenient method. In ion flotation, surface-active ions are removed from aqueous solutions by adding surfactants. Therefore, the main purpose of this review article was to summarize the application of various surfactants (nanoparticle surfactants, chemical synthetic surfactants and biosurfactants) used in ion flotation. Then, the advantages, disadvantages, and prospects of surfactants were comprehensively discussed. Recent progress regarding nanoparticle surfactants in ion flotation and the mechanism of colligends binding with nanoparticles were evaluated.
... They observed a changed response of the model in a diseased state after NP exposure in comparison to the healthy cells by performing a transcriptomic analysis [110]. This experimental setup tries to simulate the particle concentration in the atmosphere more realistically than the calculation of particle concentration in dilution (LLI) [111]. Nevertheless, complex exposure experiments are still limited, due to technical restrictions as to simulating the highest dose of airborne particles and substances compared with surrounding contaminant concentration [112]. ...
The drug development process is a lengthy and expensive challenge for all involved players. Experience with the COVID-19 pandemic underlines the need for a rapid and effective approval for treatment options. As essential prerequisites for successful drug approval, a combination of high-quality studies and reliable research must be included. To this day, mainly in vivo data are requested and collected for assessing safety and efficacy and are therefore decisive for the pre-clinical evaluation of the respective drug. This review aims to summarize the current state of the art for safety and efficacy studies in pharmaceutical research and industry to address the relevant regulatory challenges and to provide an outlook on implementing more in vitro methods as alternative to animal testing. While the public demand for alternative methods is becoming louder, first examples have meanwhile found acceptance in relevant guidelines, e.g. the OECD guidelines for skin sensitizer. Besides ethically driven developments, also the rather low throughput and relatively high costs of animal experiments are forcing the industry towards the implementation of alternative methods. In this context, the development of orally inhaled drug products is particularly challenging due to the complexity of the lung as biological barrier and route of administration. The replacement of animal experiments with focus on the lungs requires special designed tools to achieve predictive data. New in vitro test systems of increasing complexity are presented in this review. Limits and advantages are discussed to provide some perspective for a future in vitro testing strategy for orally inhaled drug products.
Graphical abstract
... The cells can form a pseudostratified airway epithelium composed of all in vivo relevant cell types found in the airway epithelium including rare types such as pulmonary neuroendocrine cells and ionocytes. 17 This type of model has proven to be particularly useful for toxicological assessment of aerosol particles, 18,19 drug discovery, 20 and more recently with the COVID-19 outbreak for viral infection studies. 21 Although these models offer significant advantages, their availability is limited to primary samples, which can significantly differ depending on the donor's genetic background and thus affecting the generated airway. ...
As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infec-tion/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs. K E Y W O R D S cytokines, induced pluripotent stem cells, interleukins (ILs), lung Significance statement Demonstration of the ability of SARS-CoV-2 to infect an airway construct generated from induced pluripotent stem cells is significant since it paves the way for broader studies of viral airway infection using a system that can be manufactured reproducibly.
... Aufderheide and Mohr 1999; Bitterle et al. 2006;Lenz et al. 2013;Lacroix et al. 2018;Tsoutsoulopoulos et al. 2019;Wang et al. 2019a;Wang et al. 2019b). These exposure systems range from those designed and used by individual research laboratories(Müller et al. 2011;Gualerzi et al. 2012;Amatngalim et al. 2015;Ji et al. 2017;Dwivedi et al. 2018) to widely available systems that are commercially marketed(Aufderheide and Mohr 1999;Okuwa et al. 2010;Li et al. 2012;Tsoutsoulopoulos et al. 2019) ...
The lung is an organ that is directly exposed to the external environment. Given the large surface area and extensive ventilation of the lung, it is prone to exposure to airborne substances, such as pathogens, allergens, chemicals, and particulate matter. Highly elaborate and effective mechanisms have evolved to protect and maintain homeostasis in the lung. Despite these sophisticated defense mechanisms, the respiratory system remains highly susceptible to environmental challenges. Because of the impact of respiratory exposure on human health and disease, there has been considerable interest in developing reliable and predictive in vitro model systems for respiratory toxicology and basic research. Human air-liquid-interface (ALI) organotypic airway tissue models derived from primary tracheobronchial epithelial cells have in vivo–like structure and functions when they are fully differentiated. The presence of the air-facing surface allows conducting in vitro exposures that mimic human respiratory exposures. Exposures can be conducted using particulates, aerosols, gases, vapors generated from volatile and semi-volatile substances, and respiratory pathogens. Toxicity data have been generated using nanomaterials, cigarette smoke, e-cigarette vapors, environmental airborne chemicals, drugs given by inhalation, and respiratory viruses and bacteria. Although toxicity evaluations using human airway ALI models require further standardization and validation, this approach shows promise in supplementing or replacing in vivo animal models for conducting research on respiratory toxicants and pathogens.
... Moreover, by suspending ENMs in cell culture media, important changes in their physicochemical properties including surface reactivity and agglomeration state occur influencing the exposure concentrations and outcome of the experiments [19,20]. An improved in vitro technique which encompasses the above-mentioned limitations is represented by air-liquid interface (ALI) exposure [21]. This allows a direct exposure to ENMs, closely mimicking realistic inhalation conditions [19,[22][23][24]. ...
Background
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.
Results
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.
Conclusion
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.
Electronic supplementary material
The online version of this article (10.1186/s12989-019-0297-1) contains supplementary material, which is available to authorized users.
... The lung is considered an important portal of entry into the human body for aerosolised toxicants smaller than 10 μm (Maynard and Kuempel, 2005;Arora et al., 2012;Nowack et al., 2012;Müller et al., 2011). Therefore, the possible effects of inhaled toxicants are of great interest in hazard and risk assessment studies. ...
Positive controls are an important feature in experimental studies as they show the responsiveness of the model under investigation. An often applied reagent for a pro-inflammatory stimulus is the endotoxin lipopolysaccharide (LPS), which has been shown to induce a cytokine release by various cell cultures.
The effect of LPS in monocultures of 16HBE14o-, a bronchial cell line, and of A549, an alveolar cell line, were compared in submerged and air-liquid interface cultures, as well as in co-cultures of the two epithelial cells with monocyte-derived macrophages and dendritic cells. The protein and mRNA levels of the two most relevant pro-inflammatory mediators, Tumor necrosis factor alpha (TNF) and Interleukin 8 (CXCL8), were measured after 4 h and 24 h exposure.
16HBE14o- cells alone as well as in co-cultures are non-responsive to an LPS stimulus, but an already increased basal expression of both pro-inflammatory mediators after prolonged time in culture was observed. In contrary, A549 in monocultures showed increased CXCL8 production at the gene and protein level after LPS exposure, while TNF-levels were below detection limit. In A549 co-cultured with immune cells both mediators were upregulated.
This study shows the importance of a careful evaluation of the culture system used, including the application of positive controls. In addition, the use of co-cultures with immune cells more adequately reflects the inflammatory response upon exposure to toxicants.
... Although it is the optimal exposure mode from a biological/toxicological standpoint, exposure at the air-liquid interface represents a considerable methodological challenge. First, it requires reliable methods for aerosol generation (e.g., smoking machines and standardized smoking protocols (Thorne and Adamson, 2013)) and application (i.e., aerosol exposure systems (Paur et al., 2011;Thorne and Adamson, 2013;Breheny et al., 2014;Muller et al., 2011;Fröhlich et al., 2013)). Second, aerosol constituents must be transferred from the aerosol into the liquid lining of a cell culture to exert an effect on the biological test system. ...
... The physical mechanisms underlying this transfer typically differ for small and large particles, for particles and volatile compounds, and for volatile compounds of different vapor pressure, polarity, and molecular mass (Pankow, 2001) (Fig. 1). In commonly used exposure systems (Thorne and Adamson, 2013;Breheny et al., 2014;Muller et al., 2011;Fröhlich et al., 2013;Paur et al., 2008), the transfer of large particles-and of chemical compounds adsorbed to or condensed on their surfaces or contained in their cores-is driven mainly by gravitation (and to a limited extent by inertial impaction). Brownian motion becomes more important with decreasing particle size (Von der Weiden et al., 2009), whereas the transfer of gaseous constituents relies primarily on diffusion (Pankow, 2001;Tippe et al., 2002;Davidovits et al., 2006). ...
In vitro aerosol exposure of epithelial cells grown at the air-liquid interface is an experimental methodology widely used in respiratory toxicology. The exposure depends to a large part on the physicochemical properties of individual aerosol constituents, as they determine the transfer kinetics from the aerosol into the cells. We characterized the transfer of 70 cigarette smoke constituents from the smoke into aqueous samples exposed in the Vitrocell® 24/48 aerosol exposure system. The amounts of these compounds in the applied smoke were determined by trapping whole smoke in N,N-Dimethylformamide and then compared with their amounts in smoke-exposed, phosphate-buffered saline, yielding compound specific delivery efficiencies. Delivery efficiencies of different smoke constituents differed by up to five orders of magnitude, which indicates that the composition of the applied smoke is not necessarily representative for the delivered smoke. Therefore, dose metrics for in vitro exposure experiments should, if possible, be based on delivered and not applied doses. A comparison to literature on in vivo smoke retention in the respiratory tract indicated that the same applies for smoke retention in the respiratory tract.
... As with our previous study, a multicellular in vitro human lung model, composed of epithelial lung cells (A549 alveolar type II-like cells) and two immune cell types (human blood monocyte-derived macrophages and dendritic cells) (Rothen-Rutishauser et al., 2005Blank et al., 2006) cultured at the air-liquid interface (ALI) (Blank et al., 2007) was used. This well-established model has been proven to be suitable for various exposures at the ALI (Fytianos et al., 2016;Müller et al., 2011), thus reflecting, in part, the realistic physiological conditions of a respiratory exposure. Further, human epithelial airway barrier models have previously accompanied hazard assessment studies of products of different engines and/or fuels using a sophisticated, well-characterised exhaust exposure system (Bisig et al., 2015Steiner et al., 2013a,b;Müller et al., 2012Müller et al., , 2011Müller et al., , 2010. ...
... This well-established model has been proven to be suitable for various exposures at the ALI (Fytianos et al., 2016;Müller et al., 2011), thus reflecting, in part, the realistic physiological conditions of a respiratory exposure. Further, human epithelial airway barrier models have previously accompanied hazard assessment studies of products of different engines and/or fuels using a sophisticated, well-characterised exhaust exposure system (Bisig et al., 2015Steiner et al., 2013a,b;Müller et al., 2012Müller et al., , 2011Müller et al., , 2010. Here, the multicellular model was directly exposed to gasoline emissions, followed by the addition of respirable volcanic ash and a second gasoline emissions exposure. ...
... The exhaust exposure experiments were performed at the exhaust gas control station of the Bern University of Applied Sciences in Nidau, Switzerland, as previously described (Bisig et al., 2015;Müller et al., 2010Müller et al., , 2011Morin et al., 2008). Briefly, the exhaust was diluted 1:10 in filtered air, based on previous work (Steiner et al., 2013a(Steiner et al., , 2013b and to enable comparison with previous gasoline exhaust studies (Bisig et al., 2015, where it was noted that it represents a highly-polluted site (i.e., a high dose exposure). ...
Communities resident in urban areas located near active volcanoes can experience volcanic ash exposures during, and following an eruption, in addition to sustained exposures to high concentrations of anthropogenic air pollutants (e.g., vehicle exhaust emissions). Inhalation of anthropogenic pollution is known to cause the onset of, or exacerbate respiratory and cardiovascular diseases. It is further postulated similar exposure to volcanic ash can also affect such disease states. Understanding of the impact of combined exposure of volcanic ash and anthropogenic pollution to human health, however, remains limited.
The aim of this study was to assess the biological impact of combined exposure to respirable volcanic ash (from Soufrière Hills volcano (SHV), Montserrat and Chaitén volcano (ChV), Chile; representing different magmatic compositions and eruption styles) and freshly-generated complete exhaust from a gasoline vehicle. A multicellular human lung model (an epithelial cell-layer composed of A549 alveolar type II-like cells complemented with human blood monocyte-derived macrophages and dendritic cells cultured at the air-liquid interface) was exposed to diluted exhaust (1:10) continuously for 6 h, followed by immediate exposure to the ash as a dry powder (0.54 ± 0.19 µg/cm2 and 0.39 ± 0.09 µg/cm2 for SHV and ChV ash, respectively). After 18 h incubation, cells were exposed again for 6 h to diluted exhaust, and a final 18-h incubation (at 37°C and 5% CO2). Cell cultures were then assessed for cytotoxic, oxidative stress and (pro-)inflammatory responses.
Results indicate that, at all tested (sub-lethal) concentrations, co-exposures with both ash samples induced no significant expression of genes associated with oxidative stress (HMOX1, NQO1) or production of (pro-)inflammatory markers (IL-1β, IL-8, TNF-α) at the gene and protein levels. In summary, considering the employed experimental conditions, combined exposure of volcanic ash and gasoline vehicle exhaust has a limited short-term biological impact to an advanced lung cell in vitro model.
... These testing strategies will require both realistic lung models and proper dosimetry techniques. The need for in vitro models predictive of inhalation toxicity and lung disease formation in humans is well recognized by the respiratory research community (Oberd€ orster et al. 2005a(Oberd€ orster et al. , 2005bSayes et al. 2007;B erub e et al. 2009;Jones & Grainger 2009;Geiser & Kreyling 2010;Huh et al. 2010;M€ uller et al. 2011;Klein et al. 2013;Herzog et al. 2014;Grabinski et al. 2015). While traditional toxicity studies in vivo have the advantage of reconstituting organ-and organism-level functions, given the costs, ethical concerns, and questionable human relevance of animal studies, it is arguable that modern in vitro testing techniques may reduce and possibly replace certain animal tests if adequate systems are developed and correlated to the in vivo human response (Sayes et al. 2007;Jones & Grainger 2009;Nel et al. 2013;Bachler et al. 2015). ...
... The most easily measured and reported dose measurement, administered (exposure) dose, in AIC exposure refers to the measurement of nanoparticle mass, surface area, or number of particles that is added to the medium (air or liquid) during exposure. Although metrics in mass ) and surface area concentrations (Bitterle et al. 2006;Raemy et al. 2011;Lenz et al. 2013) have been reported, administered dose is most commonly reported in particle number concentrations measured using scanning mobility particle sizer/condensation particle counters (SMPS/CPC) (Bitterle et al. 2006;Savi et al. 2008;de Bruijne et al. 2009;Baber et al. 2011;M€ uller et al. 2011;Raemy et al. 2012;Asimakopoulou et al. 2013;Lenz et al. 2013). Occasionally, other online particle sizers are used, such as fast mobility particle sizer (FMPS) and diffusion size classifiers like the DiscMini (M€ uller et al. 2011;Raemy et al. 2012). ...
... Although metrics in mass ) and surface area concentrations (Bitterle et al. 2006;Raemy et al. 2011;Lenz et al. 2013) have been reported, administered dose is most commonly reported in particle number concentrations measured using scanning mobility particle sizer/condensation particle counters (SMPS/CPC) (Bitterle et al. 2006;Savi et al. 2008;de Bruijne et al. 2009;Baber et al. 2011;M€ uller et al. 2011;Raemy et al. 2012;Asimakopoulou et al. 2013;Lenz et al. 2013). Occasionally, other online particle sizers are used, such as fast mobility particle sizer (FMPS) and diffusion size classifiers like the DiscMini (M€ uller et al. 2011;Raemy et al. 2012). The average background concentration of airborne particles is on the order of 10 4 particles/cm 3 or 10 lg/m 3 (Wehner et al. 2002;He et al. 2004;Paur et al. 2011). ...
Little consistency exists in the methodology for toxicological testing of aerosolized nanoparticles used in in vitro, air-interfaced culture (AIC) exposure systems for engineered nanoparticles (ENPs) risk-assessment, preventing inter-laboratory comparisons to identify dose thresholds for adverse effects. These inconsistencies result from heterogeneity in particle types, exposure durations, exposure systems, and dose metrics reported. We screened 10,241 studies in the literature for toxicological assessment of ENPs, resulting in 110 publications included after meeting eligibility criteria. In this review, we critically analyzed methodology within these studies to answer whether: (1) the administered dose or the deposited dose correlated better with biological response, (2) a difference existed between various AIC exposure systems when depositing the same dose, (3) consistent results were generated for nanomaterials with similar physico-chemical properties, (4) the deposited dose in vitro correlated to the deposited dose in vivo, and (5) AIC studies reliably modeled acute toxicity in vivo. Methods used in delivering, measuring, and reporting ENP aerosol doses in vitro are summarized. Dosimetry and biological response comparisons of AIC, conventional suspensions, and in vivo exposures are discussed through case studies on silver, zinc oxide, titanium dioxide, and multi-walled carbon nanotube exposures. Finally, based on these findings, recommendations are offered for design of future AIC experiments to aid standardization and comparisons of results.
