Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

The CULTEX (R) Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX (R) RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 mu g/cm(2)). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX (R) RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.

In vivo experiments are still widely used for the testing of lung toxicity but there is an ethical and legal obligation to replace, reduce and refine animal testing. Lung A549 cells could serve as an in vitro indicator for acute lung toxicity but little data about the correlation of the cytotoxicity in A549 cells and data leading to CLP classifications are available. We exposed A549 cells to 19 CLP-classified substances with doses of 25, 50, and 100 mu g/cm(2) either under submerged (SME) condition or with aerosols at the air-liquid interface (ALIF) and determined accuracy, precision, sensitivity and the F1 score with the CLP classifications H330, H332, or H335. When data from both exposure methods were combined, we found accuracies of 0.84 +/- 0.05, precisions of 0.74 +/- 0.1, sensitivities of 0.93 +/- 0.08 and F1 scores of 0.82 +/- 0.04. Separated from each other, ALIF exposure was more sensitive at any dose but, at higher doses, also less accurate and precise compared to SME. Considering the 19 substances tested, our data suggest that cytotoxicity in A549 cells could be a reliable in vitro indicator for in vivo toxicity. Thus, we discuss how A549 could be integrated into validation test guidelines.

Air pollution and particulate matter are recognised cause of increased disease incidence in exposed population. The toxicological processes underlying air pollution associated effects have been investigated by in vivo and/or in vitro experimentation. The latter is usually performed by exposing cells cultured under submerged condition to particulate matter concentration quite far from environmental exposure expected in humans. Here we report for the first time the feasibility of a direct exposure of air liquid interface cultured cells to environmental concentration of particulate matter. Inflammatory proteins release was analysed in cell medium while differential expression of selected genes was analysed in cells. Significant association of anti-oxidant genes was observed with secondary and aged aerosol, while cytochrome activation with primary and PAHs enriched ultrafine particles. The results obtained clearly show the opportunity to move from the lab bench to the field for properly understanding the toxicological effects also of ultrafine particles on selected in vitro models.

In February 2017 the “Carbonaceous Aerosol in Rome and Environs (CARE)” experiment was carried out in downtown Rome to address the following specific questions: what is the color, size, composition, and toxicity of the carbonaceous aerosol in the Mediterranean urban background area of Rome? The motivation of this experiment is the lack of understanding of what aerosol types are responsible for the severe risks to human health posed by particulate matter (PM) pollution, and how carbonaceous aerosols influence radiative balance. Physicochemical properties of the carbonaceous aerosol were characterised, and relevant toxicological variables assessed. The aerosol characterisation includes: (i) measurements with high time resolution (min to 1–2 h) at a fixed location of black carbon (eBC), elemental carbon (EC), organic carbon (OC), particle number size distribution (0.008–10 μ m), major non refractory PM1 components, elemental composition, wavelength-dependent optical properties, and atmospheric turbulence; (ii) 24-h measurements of PM10 and PM2.5 mass concentration, water soluble OC and brown carbon (BrC), and levoglucosan; (iii) mobile measurements of eBC and size distribution around the study area, with computational fluid dynamics modeling; (iv) characterisation of road dust emissions and their EC and OC content. The toxicological assessment includes: (i) preliminary evaluation of the potential impact of ultrafine particles on lung epithelia cells (cultured at the air liquid interface and directly exposed to particles); (ii) assessment of the oxidative stress induced by carbonaceous aerosols; (iii) assessment of particle size dependent number doses deposited in different regions of the human body; (iv) PAHs biomonitoring (from the participants into the mobile measurements). The first experimental results of the CARE experiment are presented in this paper. The objective here is to provide baseline levels of carbonaceous aerosols for Rome, and to address future research directions. First, we found that BC and EC mass concentration in Rome are larger than those measured in similar urban areas across Europe (the urban background mass concentration of eBC in Rome in winter being on average 2.6 ± 2.5 μ g · m − 3 , mean eBC at the peak level hour being 5.2 (95% CI = 5.0–5.5) μ g · m − 3 ). Then, we discussed significant variations of carbonaceous aerosol properties occurring with time scales of minutes, and questioned on the data averaging period used in current air quality standard for PM 10 (24-h). Third, we showed that the oxidative potential induced by aerosol depends on particle size and composition, the effects of toxicity being higher with lower mass concentrations and smaller particle size. Albeit this is a preliminary analysis, findings reinforce the need for an urgent update of existing air quality standards for PM 10 and PM 2.5 with regard to particle composition and size distribution, and data averaging period. Our results reinforce existing concerns about the toxicity of carbonaceous aerosols, support the existing evidence indicating that particle size distribution and composition may play a role in the generation of this toxicity, and remark the need to consider a shorter averaging period (<1 h) in these new standards.

This paper explores the possibility of using subway maps as a planning tool to influence passenger route choice to mitigate congestion. Specifically, it tests whether extending the appearance of an overcrowded subway line on the Washington DC subway map would encourage passengers to use other underutilized lines. The experiment was conducted through the Mechanical Turk, a crowdsourcing platform, with 3056 participants, producing 21,240 route choice decisions on the official and six alternative maps. Results show that redesigned maps significantly affect participants’ route choices. Depending on the specific design, a 20% length increase of the overcrowded line could move 1.9–5.7 percentage points of ridership to an alternative, underutilized line. The change could remove up to 10 passengers per car during the highest peak, reducing the number of highly congested half-hour periods (max load = 100–120 passengers per car) on the overcrowded line from 4 to 1, and the number of crush periods (max load > 120 passengers per car) from 3 to 1. This is done at minimal or zero cost. The paper calls for more attention from transit agencies to the planning potential of transit maps.

The exposure of cellular based systems cultivated on microporous membranes at the air-liquid interface (ALI) has been accepted as an appropriate approach to simulate the exposure of cells of the respiratory tract to native airborne substances. The efficiency of such an exposure procedure with regard to stability and reproducibility depends on the optimal design at the interface between the cellular test system and the exposure technique. The actual exposure systems favor the dynamic guidance of the airborne substances to the surface of the cells in specially designed exposure devices. Two module types, based on a linear or radial feed of the test atmosphere to the test system, were used for these studies. In our technical history, the development started with the linear designed version, the CULTEX® glass modules, fulfilling basic requirements for running ALI exposure studies (Mohr and Durst, 2005). The instability in the distribution of different atmospheres to the cells caused us to create a new exposure module, characterized by a stable and reproducible radial guidance of the aerosol to the cells. The outcome was the CULTEX® RFS (Mohr et al., 2010). In this study, we describe the differences between the two systems with regard to particle distribution and deposition clarifying the advantages and disadvantages of a radial to a linear aerosol distribution concept.

3D constructs composed of primary normal differentiated human bronchiolar epithelial (NHBE) cells as mono- or co-culture in combination with normal human lung fibroblasts were exposed repeatedly at the air-liquid interface with non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5days/week, 13 times repetition in total) to build up a permanent burden on the cells. Samples were taken after 4, 8 and 13 times of repeated smoke exposure and the cultures were analyzed by histopathological methods In comparison with the clean air exposure (process control) and incubator control cells the cigarette smoke exposed cultures showed a reduction of cilia bearing as well as mucus producing cells. In both mono- as well as co-cultures, hyperplasia was induced showing different histological cell types (undifferentiated secretory and squamous cell types). At the end of the exposure phase, we observed the development of non-hyperplastic areas strongly positive to CK13 antibody, commonly seen in squamous cells as a marker for non-cornified squamous epithelium, thus suggesting a transition of the normal bronchial epithelial cells towards metaplastic cells. The control cultures (clean air exposed and incubator cells) showed no comparable phenotypic changes. In conclusion, our in vitro model presents a valuable tool to study the induction of metaplastic alterations after exposure to airborne material.

3D constructs composed of differentiated immortalized primary normal human bronchial epithelial (NHBE) cells (CL-1548) were repeatedly exposed at the air-liquid interface to non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5 days/week, 8 repetitions in total) and e-cigarette vapor (50 puffs a day, 5 days/week, 8 repetitions in total) to build up a permanent burden on the cells. Samples were taken after 4, 6 and 8 times of repeated smoke exposure and the cultures were investigated using histopathological methods Compared to the clean air-exposed cultures (process control) and incubator control, the aerosol-exposed cultures showed a reduction of ciliated, mucus-producing and club cells. At the end of the exposure phase, we even found metaplastic areas positive for CK13 antibody in the cultures exposed to mainstream cigarette smoke and e-liquid vapor, commonly seen in squamous cells as a marker for non-cornified squamous epithelium. The control cultures (incubator cells) showed no comparable phenotypicalchanges. In conclusion, our in vitro model presents a valuable tool to study the induction of phenotypical changes after exposure to hazardous airborne material.

Exposure of the respiratory tract to airborne particles is gaining more and more importance due to the ubiquitous application of these particles in the field of industry, pharmacy and in daily life. Remarkably, the toxic properties and the underlying pathomechanisms with regard to inhalable substances have been insufficiently investigated so far. Thus, the EU Chemicals Regulation demands toxicological data (including the identification of potential inhalation hazards) for all chemicals placed on the market until 2018 (REACH). This requires extensive, technically complex and expensive inhalation toxicology studies that are usually generated in animal experiments. However, the legislation demands the consideration of the "3Rs" principle. Thus, in vitro-based test systems for the assessment of pulmonary toxicity are required. One promising approach to assess acute pulmonary toxicity of airborne particles is the CULTEX(®) RFS methodology that allows exposure of human lung epithelial cells at the air-liquid interface mimicking the alveolar situation. A prevalidation study showed the general applicability of this method. However, the clean air exposure group, which served as unexposed controls, exhibited some variations with regard to cell viability compared to the incubator control group. The aim of this study was therefore the identification of the possible causes and the improvement of methodological aspects. Several parameters including the general workflow, adjustment of airflow parameters, and cleaning procedures were investigated and adapted. Finally, our results showed the successful optimization of the CULTEX(®) RFS methodology for clean air exposure of A549 cells. However, although viability data in incubator controls and clean air exposures were equal, a distinct difference in cell morphology was observed that required further optimization. Additional experiments identified that open-wall cell culture inserts with a 2-fold pore density were found to be superior compared to the standard inserts and thus the deciding factor for the improvement of cell morphology. The presented findings are an important step in providing the CULTEX(®) RFS methodology as a promising alternative method to current in vivo testing in inhalation toxicology.

There is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or electronic cigarettes (ECs) aerosol emissions in one single testing. However, there is growing confusion, with several researchers endorsing their personal solution to the problem. Here, we discuss as an example the recent paper by Scheffler and colleagues, in which the authors suggest that more relevant and well differentiated cell lines from human airways could be the most suitable candidates for toxicological evaluation of ECs aerosol emissions. We advance recommendations for validated protocols and advocate for an international coordinated effort aimed at establishing consensus on methodology.

The in vitro toxicological evaluation of e-liquid aerosol is an important aspect of consumer protection, but the cell model is of great significance. Due to its water solubility, e-liquid aerosol is deposited in the conducting zone of the respiratory tract. Therefore, primary normal human bronchial epithelial (NHBE) cells are more suitable for e-liquid aerosol testing than the widely used alveolar cell line A549. Due to their prolonged lifespan, immortalized cell lines derived from primary NHBE cells, exhibiting a comparable in vitro differentiation, might be an alternative for acute toxicity testing. In our study, A549 cells freshly isolated NHBE cells and the immortalized cell line CL-1548 were exposed at the air-liquid interface to e-liquid aerosol and cigarette mainstream smoke in a CULTEX® RFS compact module. The cell viability was analyzed 24 h post-exposure. In comparison with primary NHBE cells, the CL-1548 cell line showed lower sensitivity to e-liquid aerosol but significantly higher sensitivity compared to A549 cells. Therefore, the immortalized cell line CL-1548 is recommended as a tool for the routine testing of e-liquid aerosol and is preferable to A549 cells.

Mucociliary clearance is the primary physical mechanism to protect the human airways against harmful effects of inhaled particles. Environmental factors play a significant role in the impairment of this defense mechanism, whereas cigarette smoke is discussed to be one of the clinically most important causes. Impaired mucociliary clearance in smokers has been connected to changes in ciliated cells such as decreased numbers, altered structure and beat frequency. Clinical studies have shown that cilia length is reduced in healthy smokers and that long-term exposure to cigarette smoke leads to reduced numbers of ciliated cells in mice. We present an in vitro model of primary normal human bronchiolar epithelial (NHBE) cells with in vivo like morphology to study the influence of cigarette mainstream smoke on ciliated cells. We exposed mucociliary differentiated cultures repeatedly to non-toxic concentrations of mainstream cigarette smoke (4 cigarettes, 5 days/week, 8 repetitions in total) at the air-liquid interface. Charcoal filter tipped cigarettes were compared to those being equipped with standard cellulose acetate filters. Histopathological analyses of the exposed cultures showed a reduction of cilia bearing cells, shortening of existing cilia and finally disappearance of all cilia in cigarette smoke exposed cells. In cultures exposed to charcoal filtered cigarette smoke, little changes in cilia length were seen after four exposure repetitions, but those effects were reversed after a two day recovery period. Those differences indicate that volatile organic compounds, being removed by the charcoal filter tip, affect primary bronchiolar epithelial cells concerning their cilia formation and function comparable with the in vivo situation. In conclusion, our in vitro model presents a valuable tool to study air-borne ciliatoxic compounds.

E-cigarettes are emerging products, often described as “reduced-risk” nicotine products or alternatives to combustible cigarettes. Many smokers switch to e-cigarettes to quit or significantly reduce smoking. However, no regulations for e-cigarettes are currently into force, so that the quality and safety of e-liquids is not necessarily guaranteed. We exposed primary human bronchial epithelial cells of two different donors to vapor of e-cigarette liquid with or without nicotine, vapor of the carrier substances propylene glycol and glycerol as well as to mainstream smoke of K3R4F research cigarettes. The exposure was done in a CULTEX® RFS compact module, allowing the exposure of the cells at the air-liquid interface. 24 h post-exposure, cell viability and oxidative stress levels in the cells were analyzed. We found toxicological effects of e-cigarette vapor and the pure carrier substances, whereas the nicotine concentration did not have an effect on the cell viability. The viability of mainstream smoke cigarette exposed cells was 4.5–8 times lower and the oxidative stress levels 4.5–5 times higher than those of e-cigarette vapor exposed cells, depending on the donor. Our experimental setup delivered reproducible data and thus provides the opportunity for routine testing of e-cigarette liquids to ensure safety and quality for the user.

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable, reproducible and controllable cultivation conditions. Up to now, some devices have been developed for the cultivation of cells under submersed conditions. However, systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose, a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts), which can be adjusted for the medium level (ultrasonic pulse-echosensor), time and volume-dependent medium exchange, and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells.

Recent advancement in research on stem/progenitor cells of respiratory organs is breathtaking, benefiting from the rapid development of technology to create transgenic mice. There is now a great deal of knowledge capable of direct translation from mice to humans. Nevertheless, one has to be careful, since there may be unexpected pitfalls. First of all, there are differences anatomically, histologically and ultrastructurally in the airway epithelia of the two species. In parallel with these structural differences, regionally specific cell types behave and function, particularly in regenerative instances, differently between the two species, at least to some extent. From the viewpoint of important human respiratory diseases, one of the most susceptible regions of the respiratory tract is the bronchiole. In our approach to develop in vitro systems utilizing human bronchiolar epithelial cells, we are currently leaning on the data obtained from mouse studies in spite of the above-mentioned species differences. With the help of such in vitro systems we should be able to investigate the damaging effects and mechanisms of environmental pollutants in the human respiratory epithelium and consequently achieve results useful for quantitative analyses of the impact on human respiratory health. While pursuing this goal, the mouse data have suggested that it should be worthwhile to pay close attention to the stem/progenitor cells contained in the human bronchiolar epithelia and eventually make use of them. The mouse data have further shown that these stem/progenitor cells possess a very close association with the immature and variant club cells and the neuroendocrine cells, and our own unpublished preliminary data with human cells are, apparently, at least partly consistent with what the mouse data are telling us.

In toxicology, the strategies for testing the hazardous potential of substances are changing as a result of the ongoing progress in the development of in vitro methods and the demand of the authorities to reduce animal testing. Even in the complex field of inhalation toxicology with its high requirements on the technical implementation and cell culture models, the preconditions for using such methods are fulfilled. We here introduce a sophisticated technique that enables the stable and reproducible exposure of cultivated cells to airborne substances at the air-liquid interface by means of the CULTEX(®) Radial Flow System (RFS) module. The feasibility and suitability of the experimental setup is demonstrated by dose-response investigations of mainstream cigarette smoke and particulate matter of four substances in different lung epithelial cell lines. A dose-dependent cytotoxcity of the test substances was verified by applying different exposure times. The high reproducibility of the results indicate the reliability of the presented method and recommend the integration of such in vitro approaches in the field of inhalation toxicology by advancing their regulatory validation.

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general (“3R” principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials.Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air–liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility.Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results.Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.

In vitro approaches, especially cell culture techniques, have gained more and more importance in toxicology during the last decade, due to the ongoing progress in the development of novel technologies and the increasing call for alternatives to animal testing. One major requirement for the reliability of toxicological data is their reproducibility. For that purpose, identical cell cultures are required for repeatable experiments. However, the handling of cell cultures is laborious and vulnerable to personspecific variations as well as pipetting errors. Thus, automated cell cultivation systems are highly desirable for in vitro toxicology. There are various automated systems that are specifically designed to fulfill particular requirements, ranging from different bioreactors up to sophisticated modular working stations.