A newly developed in vitro model of the human epithelial airway barrier to study the toxic potential of nanoparticles.
ABSTRACT 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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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 · 7.34 Impact Factor
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ABSTRACT: Epithelia act in the organism as biological barriers. All of them are exposed to different environments at the luminal and basal side. To simulate such a tissue-specific situation Minusheet ® gradient perfusion culture was developed. For pharmaceutical research, biomaterial testing and tissue engineering epithelial cells are cultured on individually se-lected supports (1). Growing epithelia are stabilized within a tissue carrier (2). Long term culture is performed in a gradi-ent perfusion container (3). To expose epithelia to a tissue-specific environment fresh media of different composition are transported parallel to the luminal and basal compartment of the gradient container. During culture leakage, edge damage and pressure differences have to be avoided. Harvest of intact epithelia is promoted by the use of biocompatible supports and innovative equipment such as transport of oxygen-rich and gas bubble-free medium. Actual literature demonstrates that gradient perfusion culture is an effective method to investigate barrier functions under realistic conditions. Examples of application comprise renal epithelia, retina, blood-air barrier, blood-brain barrier including aspects of tissue-specific development and regeneration. Beside the nervous tissue, the muscular tissue and the connective tissue epithelia belong to the fourth group of ba-sic tissues in the organism. All of the epithelia exhibit impor-tant barrier functions. They are heterogeneously composed, consist as simple, pseudostratified or stratified epithelia and contain squamous, cuboidal or columnar cells. The tasks of epithelia are manifold. The protection of underlying tissues is the role of epithelium covering the external surfaces and orifices, while transport of mucus and particles is performed by ciliated epithelia found in secretory, respiratory and geni-tal ducts. The epithelia of the intestine, liver and kidney are involved in absorption, secretion and filtering of molecules from and into a lumen. In follicles of the thyroid gland and ovary the relation of the epithelia to a free surface is re-tained. Taste buds and olfactory mucosa epithelia are in-volved in sensory reception. Typical for all of the epithelia is that they rest on a layer of extracellular matrix called base-ment membrane or basal lamina.Journal of Epithelial Biology & Pharmacology 04/2009; 2(1). DOI:10.2174/1875044300902010001
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ABSTRACT: Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.Journal of The Royal Society Interface 08/2009; 7 Suppl 1:S27-40. DOI:10.1098/rsif.2009.0161.focus · 3.86 Impact Factor