A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles

Institute of Anatomy, Division of Histology, University of Bern, Bern, Switzerland.
ALTEX 02/2008; 25(3):191-6.
Source: PubMed


The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway. Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha. Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

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    • "Therefore, the purpose of this study was to develop a 3D intestinal co-culture model composed of cell lines, which mimics healthy and diseased conditions and is suitable to assess NP-cell interactions. Co-culture models have been widely accepted (Rothen- Rutishauser et al., 2005, 2008) and used in several studies, e.g. to evaluate NP toxicity when exposed to the lungs by inhalation (Muller et al., 2010; Rothen-Rutishauser et al., 2008). Regarding the intestinal epithelium, i.e. after oral ingestion, most of the models focus on co-cultures to study absorption, particle celltranslocation or particle-mucus interaction (Schimpel et al., 2014). "
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    ABSTRACT: Abstract Oral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO2, NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1β, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO2 or Au NPs. But once inflamed with IL-1β, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation.
    Nanotoxicology 03/2015; DOI:10.3109/17435390.2015.1008065 · 6.41 Impact Factor
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    • "Since there are many efforts on-going to use sophisticated in vitro methods for toxicology testing in order to reduce the number of invasive animal-based testing strategies [37] our research group has established and evaluated an in vitro model of the human epithelial airway barrier composed of epithelial cells and the two most important immune cells of the lung (i.e. macrophages and dendritic cells), to study NP lung-cell interactions and their possible responses [38]. Since this model can be used at the air-liquid interface it allows the direct exposure of cells to an aerosol [39], thus representing a realistic situation following inhalation of NPs. "
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    ABSTRACT: Background Due to its antibacterial properties, silver (Ag) has been used in more consumer products than any other nanomaterial so far. Despite the promising advantages posed by using Ag-nanoparticles (NPs), their interaction with mammalian systems is currently not fully understood. An exposure route via inhalation is of primary concern for humans in an occupational setting. Aim of this study was therefore to investigate the potential adverse effects of aerosolised Ag-NPs using a human epithelial airway barrier model composed of A549, monocyte derived macrophage and dendritic cells cultured in vitro at the air-liquid interface. Cell cultures were exposed to 20 nm citrate-coated Ag-NPs with a deposition of 30 and 278 ng/cm2 respectively and incubated for 4 h and 24 h. To elucidate whether any effects of Ag-NPs are due to ionic effects, Ag-Nitrate (AgNO3) solutions were aerosolised at the same molecular mass concentrations. Results Agglomerates of Ag-NPs were detected at 24 h post exposure in vesicular structures inside cells but the cellular integrity was not impaired upon Ag-NP exposures. Minimal cytotoxicity, by measuring the release of lactate dehydrogenase, could only be detected following a higher concentrated AgNO3-solution. A release of pro-inflammatory markers TNF-α and IL-8 was neither observed upon Ag-NP and AgNO3 exposures as well as was not affected when cells were pre-stimulated with lipopolysaccharide (LPS). Also, an induction of mRNA expression of TNF-α and IL-8, could only be observed for the highest AgNO3 concentration alone or even significantly increased when pre-stimulated with LPS after 4 h. However, this effect disappeared after 24 h. Furthermore, oxidative stress markers (HMOX-1, SOD-1) were expressed after 4 h in a concentration dependent manner following AgNO3 exposures only. Conclusions With an experimental setup reflecting physiological exposure conditions in the human lung more realistic, the present study indicates that Ag-NPs do not cause adverse effects and cells were only sensitive to high Ag-ion concentrations. Chronic exposure scenarios however, are needed to reveal further insight into the fate of Ag-NPs after deposition and cell interactions.
    Particle and Fibre Toxicology 04/2013; 10(1):11. DOI:10.1186/1743-8977-10-11 · 7.11 Impact Factor
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    • "After the triple cell coculture was established, cell densities of macrophages and dendritic cells within the culture were quantified using the specific surface markers CD14 and CD86 for the labeling of macrophages and dendritic cells, respectively, and the quantitative occurrence of macrophages and dendritic cells resembled very closely the in-vivo situation (Blank et al., 2007). After its thorough evaluation, this model was exposed to particles (either airborne or suspended in medium) of different materials (polystyrene, titanium dioxide and gold) and of different sizes ( r1 mm) (Blank et al., 2007; Rothen- Rutishauser, 2007; Rothen-Rutishauser et al., 2008b; Brandenberger et al., 2010). Translocation and cellular localization of particles were studied as well as the effects of particles on cellular interplay and signaling. "
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    ABSTRACT: The introduction of engineered nanostructured materials into a rapidly increasing number of industrial and consumer products will result in enhanced exposure to engineered nanoparticles. Workplace exposure has been identified as the most likely source of uncontrolled inhalation of engineered aerosolized nanoparticles, but release of engineered nanoparticles may occur at any stage of the lifecycle of (consumer) products. The dynamic development of nanomaterials with possibly unknown toxicological effects poses a challenge for the assessment of nanoparticle induced toxicity and safety.In this consensus document from a workshop on in-vitro cell systems for nanoparticle toxicity testing1 an overview is given of the main issues concerning exposure to airborne nanoparticles, lung physiology, biological mechanisms of (adverse) action, in-vitro cell exposure systems, realistic tissue doses, risk assessment and social aspects of nanotechnology. The workshop participants recognized the large potential of in-vitro cell exposure systems for reliable, high-throughput screening of nanoparticle toxicity. For the investigation of lung toxicity, a strong preference was expressed for air–liquid interface (ALI) cell exposure systems (rather than submerged cell exposure systems) as they more closely resemble in-vivo conditions in the lungs and they allow for unaltered and dosimetrically accurate delivery of aerosolized nanoparticles to the cells. An important aspect, which is frequently overlooked, is the comparison of typically used in-vitro dose levels with realistic in-vivo nanoparticle doses in the lung. If we consider average ambient urban exposure and occupational exposure at 5 mg/m3 (maximum level allowed by Occupational Safety and Health Administration (OSHA)) as the boundaries of human exposure, the corresponding upper-limit range of nanoparticle flux delivered to the lung tissue is 3×10−5–5×10-3 μg/h/cm2 of lung tissue and 2–300 particles/h/(epithelial) cell. This range can be easily matched and even exceeded by almost all currently available cell exposure systems.The consensus statement includes a set of recommendations for conducting in-vitro cell exposure studies with pulmonary cell systems and identifies urgent needs for future development. As these issues are crucial for the introduction of safe nanomaterials into the marketplace and the living environment, they deserve more attention and more interaction between biologists and aerosol scientists. The members of the workshop believe that further advances in in-vitro cell exposure studies would be greatly facilitated by a more active role of the aerosol scientists. The technical know-how for developing and running ALI in-vitro exposure systems is available in the aerosol community and at the same time biologists/toxicologists are required for proper assessment of the biological impact of nanoparticles.Highlights► Air–liquid interface (ALI) cell systems facilitate pulmonary nanotoxicity testing. ► Overview on aerosol and biology issues relevant for ALI cell exposure systems. ► Upper limits for realistic nanoparticle doses delivered to the lung tissue. ► Most ALI exposure systems match or exceed realistic nanoparticle tissue doses. ► Recommendations for conducting pulmonary in-vitro cell exposure studies.
    Journal of Aerosol Science 10/2011; 42(10):668-692. DOI:10.1016/j.jaerosci.2011.06.005 · 2.24 Impact Factor
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