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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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Sodium fluorescein leakage for mono-, co-, and tetraculture in transwell inserts with 0.4, 1 and 3 μm pore size. Medium containing 10 µg/mL of sodium fluorescein was administered to the apical compartment. Medium w/o sodium fluorescein was administered to the basolateral compartment and the leaked fluorescence was measured after 60 minutes incubation at room temperature in the dark. Results obtained for inserts with a pore size of 0.4 μm, 1 μm and 3 μm are given in A, B and C, respectively. The dotted line represents the leakage of transwell inserts w/o cells. Data represents the mean of four independent transwell inserts ± SEM. Groups that are sharing the same letters are not significantly different (P > 0.05).
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Figure 5: Sodium fluorescein leakage for mono-, co-, and tetraculture in transwell inserts with 0.4, 1 and 3 μm pore size. Medium containing 10 µg/mL of sodium fluorescein was administered to the apical compartment. Medium w/o sodium fluorescein was administered to the basolateral compartment and the leaked fluorescence was measured after 60 minutes incubation at room temperature in the dark. Results obtained for inserts with a pore size of 0.4 μm, 1 μm and 3 μm are given in A, B and C, respectively. The dotted line represents the leakage of transwell inserts w/o cells. Data represents the mean of four independent transwell inserts ± SEM. Groups that are sharing the same letters are not significantly different (P > 0.05).

Mentions: A549 in monoculture, EA.hy 926 in monoculture, A549 plus EA.hy 926 in coculture and the tretraculture were exposed for 60 minutes to a fluorescent tracer solution in the apical compartment and the fluorescence leakage was measured in the basolateral compartment. Inserts of different pore sizes (0.4, 1 and 3 μm) were compared as well (Figure 5A, B and C). The 0.4 μm inserts showed a great ability to block the fluorescent tracer solution, regardless of the presence or absence of cells (Figure 5A). In inserts with 1 and 3 μm pore size, the presence and the composition of the cell layers influences the amount of leaked fluorescence. The overall leakage in an empty 0.4 μm transwell insert (w/o cells) was four times less compared to inserts with 1 and 3 μm pores (compare empty inserts in Figure 5A, B, and C). For cell cultures prepared in 0.4 μm inserts, no statistically significant differences were observed (Figure 5A). When 1 μm inserts are used, the monoculture of EA.hy 926 shows with 9103 ± 545 significantly the highest leakage (P < 0.05). When A549 and EA.hy 926 cells are in coculture, the leakage (4944 ± 93) is slightly lower than in the A549 monoculture (5241 ± 161). In the tetraculture system with A549, EA.hy 926, differentiated THP-1 and HMC-1 cells, the leakage is with 6339 ± 249 again higher than for the A549 monoculture and the coculture of A549 and EA.hy 926 but still significantly lower than for the monoculture of EA.hy 926 (P < 0.05) (Figure 5B). In transwell inserts with 3 μm pores, the amount of leaked fluorescence for A549 monoculture is 4928 ± 57 and for EA.hy 926 in monoculture 6134 ± 572. The coculture of A549 and EA.hy 926 cells shows a fluorescence leakage of 5250 ± 84. The differences between A549 and EA.hy 926 cells in monoculture and the corresponding coculture were not significant. In these inserts the tetraculture shows the highest amount of leaked fluorescence with 11584 ± 865 (Figure 5C).


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)

Sodium fluorescein leakage for mono-, co-, and tetraculture in transwell inserts with 0.4, 1 and 3 μm pore size. Medium containing 10 µg/mL of sodium fluorescein was administered to the apical compartment. Medium w/o sodium fluorescein was administered to the basolateral compartment and the leaked fluorescence was measured after 60 minutes incubation at room temperature in the dark. Results obtained for inserts with a pore size of 0.4 μm, 1 μm and 3 μm are given in A, B and C, respectively. The dotted line represents the leakage of transwell inserts w/o cells. Data represents the mean of four independent transwell inserts ± SEM. Groups that are sharing the same letters are not significantly different (P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Sodium fluorescein leakage for mono-, co-, and tetraculture in transwell inserts with 0.4, 1 and 3 μm pore size. Medium containing 10 µg/mL of sodium fluorescein was administered to the apical compartment. Medium w/o sodium fluorescein was administered to the basolateral compartment and the leaked fluorescence was measured after 60 minutes incubation at room temperature in the dark. Results obtained for inserts with a pore size of 0.4 μm, 1 μm and 3 μm are given in A, B and C, respectively. The dotted line represents the leakage of transwell inserts w/o cells. Data represents the mean of four independent transwell inserts ± SEM. Groups that are sharing the same letters are not significantly different (P > 0.05).
Mentions: A549 in monoculture, EA.hy 926 in monoculture, A549 plus EA.hy 926 in coculture and the tretraculture were exposed for 60 minutes to a fluorescent tracer solution in the apical compartment and the fluorescence leakage was measured in the basolateral compartment. Inserts of different pore sizes (0.4, 1 and 3 μm) were compared as well (Figure 5A, B and C). The 0.4 μm inserts showed a great ability to block the fluorescent tracer solution, regardless of the presence or absence of cells (Figure 5A). In inserts with 1 and 3 μm pore size, the presence and the composition of the cell layers influences the amount of leaked fluorescence. The overall leakage in an empty 0.4 μm transwell insert (w/o cells) was four times less compared to inserts with 1 and 3 μm pores (compare empty inserts in Figure 5A, B, and C). For cell cultures prepared in 0.4 μm inserts, no statistically significant differences were observed (Figure 5A). When 1 μm inserts are used, the monoculture of EA.hy 926 shows with 9103 ± 545 significantly the highest leakage (P < 0.05). When A549 and EA.hy 926 cells are in coculture, the leakage (4944 ± 93) is slightly lower than in the A549 monoculture (5241 ± 161). In the tetraculture system with A549, EA.hy 926, differentiated THP-1 and HMC-1 cells, the leakage is with 6339 ± 249 again higher than for the A549 monoculture and the coculture of A549 and EA.hy 926 but still significantly lower than for the monoculture of EA.hy 926 (P < 0.05) (Figure 5B). In transwell inserts with 3 μm pores, the amount of leaked fluorescence for A549 monoculture is 4928 ± 57 and for EA.hy 926 in monoculture 6134 ± 572. The coculture of A549 and EA.hy 926 cells shows a fluorescence leakage of 5250 ± 84. The differences between A549 and EA.hy 926 cells in monoculture and the corresponding coculture were not significant. In these inserts the tetraculture shows the highest amount of leaked fluorescence with 11584 ± 865 (Figure 5C).

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