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An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.

Klein SG, Serchi T, Hoffmann L, Blömeke B, Gutleb AC - Part Fibre Toxicol (2013)

Bottom Line: Macrophage-like cells and mast cells can be found on top of the epithelial cells.Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures.The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane.

Results: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only.

Conclusion: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

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Z-stack image series to analyse the distribution of THP-1 macrophages and HMC-1 in the tetraculture system present in the apical compartment of the insert. The distribution of A549, differentiated THP-1, HMC-1 and EA.hy 926 cells in the tetraculture was analysed via CLSM. Cellular membranes are stained with cell mask deep red dye (red) and nuclei are stained with DAPI (blue); Macrophage-like cells are counterstained with an anti-CD11b-antibody. A: X–y projection with the respective side views. B: 3D reconstruction of the tetraculture based on the results of the z-stack from A. THP-1 (green arrows) and HMC-1 (blue arrows) cells are found on top of the epithelial cells. EA.hy 926 cells were not considered in the 3D reconstruction.
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Figure 3: Z-stack image series to analyse the distribution of THP-1 macrophages and HMC-1 in the tetraculture system present in the apical compartment of the insert. The distribution of A549, differentiated THP-1, HMC-1 and EA.hy 926 cells in the tetraculture was analysed via CLSM. Cellular membranes are stained with cell mask deep red dye (red) and nuclei are stained with DAPI (blue); Macrophage-like cells are counterstained with an anti-CD11b-antibody. A: X–y projection with the respective side views. B: 3D reconstruction of the tetraculture based on the results of the z-stack from A. THP-1 (green arrows) and HMC-1 (blue arrows) cells are found on top of the epithelial cells. EA.hy 926 cells were not considered in the 3D reconstruction.

Mentions: A similar observation was made in the tetraculture system, composed of A549, differentiated THP-1, HMC-1, and EA.hy 926 cells. After seeding the HMC-1 cells into the tetraculture system, the originally floating HMC-1 cells disappeared from the culture medium and attached to the epithelial cell layer. The macrophage-like cells and mast cells can be found on top of the epithelial cells (Figure 3A and B). THP-1 cells are in direct contact with HMC-1 cells and they form heterogeneous colonies (Figure 3B). However, once the system was shifted to ALI conditions, the colonies that were observed under submerged conditions disappeared and the cells spread more equally on top of the epithelial cells (Figure 4A and B).


An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.

Klein SG, Serchi T, Hoffmann L, Blömeke B, Gutleb AC - Part Fibre Toxicol (2013)

Z-stack image series to analyse the distribution of THP-1 macrophages and HMC-1 in the tetraculture system present in the apical compartment of the insert. The distribution of A549, differentiated THP-1, HMC-1 and EA.hy 926 cells in the tetraculture was analysed via CLSM. Cellular membranes are stained with cell mask deep red dye (red) and nuclei are stained with DAPI (blue); Macrophage-like cells are counterstained with an anti-CD11b-antibody. A: X–y projection with the respective side views. B: 3D reconstruction of the tetraculture based on the results of the z-stack from A. THP-1 (green arrows) and HMC-1 (blue arrows) cells are found on top of the epithelial cells. EA.hy 926 cells were not considered in the 3D reconstruction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733942&req=5

Figure 3: Z-stack image series to analyse the distribution of THP-1 macrophages and HMC-1 in the tetraculture system present in the apical compartment of the insert. The distribution of A549, differentiated THP-1, HMC-1 and EA.hy 926 cells in the tetraculture was analysed via CLSM. Cellular membranes are stained with cell mask deep red dye (red) and nuclei are stained with DAPI (blue); Macrophage-like cells are counterstained with an anti-CD11b-antibody. A: X–y projection with the respective side views. B: 3D reconstruction of the tetraculture based on the results of the z-stack from A. THP-1 (green arrows) and HMC-1 (blue arrows) cells are found on top of the epithelial cells. EA.hy 926 cells were not considered in the 3D reconstruction.
Mentions: A similar observation was made in the tetraculture system, composed of A549, differentiated THP-1, HMC-1, and EA.hy 926 cells. After seeding the HMC-1 cells into the tetraculture system, the originally floating HMC-1 cells disappeared from the culture medium and attached to the epithelial cell layer. The macrophage-like cells and mast cells can be found on top of the epithelial cells (Figure 3A and B). THP-1 cells are in direct contact with HMC-1 cells and they form heterogeneous colonies (Figure 3B). However, once the system was shifted to ALI conditions, the colonies that were observed under submerged conditions disappeared and the cells spread more equally on top of the epithelial cells (Figure 4A and B).

Bottom Line: Macrophage-like cells and mast cells can be found on top of the epithelial cells.Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures.The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane.

Results: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only.

Conclusion: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

Show MeSH
Related in: MedlinePlus