S 3 and 7 at any time-point. However, PPS at 200 mg/ml induced strong activation already after 4 hours, reaching the maximum of an about 50-fold increase after 8 hours and an almost 30-fold increase after 24 hours as compared to non-exposed cells (Fig. 2 A). A similar dose-dependent effect was observed for membrane disruption (Fig. 2 C). Exposure of the cells to 200 nm PPSresulted in a very low release of LDH (up to 15 of the lysis control) and no notable increase in caspase activation at any AKT inhibitor 2 timepoint (Fig. 2 B and D).Establishment of the order 1113-59-3 microcarrier cell culture systemFor optimization of the exposure system, different coatings of the GEMTM (gelatine, laminin, fibronectin, collagen type I and IV, basal membrane) as well as different incubation protocols, predefined by the supplier, were compared. Cells that were seeded onFigure. 1. Acute cytotoxicity of NPs exerted on EAhy 926 23727046 in different cell cultures after 24 hours. Cells in conventional cultures were treated with NPs dissolved in serum-free medium (A) as well as in medium with 10 FBS (B). Cells cultured on microcarriers were exposed to NMs dissolved in medium with 10 FBS. Data are presented as mean 6 SD. doi:10.1371/journal.pone.0056791.gLong-Term Effects of NanoparticlesFigure. 2. Mode of action of PPS in conventional cultures. Activation of caspases 3 and 7 (A and B) and release of LDH (C and D) upon exposure of EAhy 926 cells to 20 and 200 nm PPS for 4, 8, and 24 hours compared to untreated cells. Data are presented as mean 6 SD. (h), hours. doi:10.1371/journal.pone.0056791.gbasal membrane coated microcarriers and cultured according to the protocol for endothelial cells (HUVEC) reached approximately 4 times higher cell densities compared to those when the protocol for epithelial cells (HEK 293) was used (Fig. 3). Cultures were maintained for 23 days without sub-culturing. The doubling time of EAhy cells was lower in the microcarrier culture system (70 h) than in conventional cultures (30 h). Staining with Hoechst 33342 dye showed an increase in cell proliferation on GEMTM (Fig. 4 A). Furthermore, vital staining for mitochondria and endoplasmic reticulum (ER) demonstrated the viability of the cells (Fig. 4 B).Long-term effects of NPs in microcarrier cultureCells were cultured according to the established protocol (basal membrane coated GEMTM, and incubation protocol for endothelial cells) for four weeks with a medium change performed once per week. After inoculation, NPs were added at a concentration 250 times lower than the concentration where cytotoxicity was seen in the acute cytotoxicity setting (24 hours). Exposure of the cells to 20 mg/ml of 20 nm PPS resulted in a significantly reduced cell number already after 7 days, showing a decrease in cell numbers of approximately 50 . Even stronger effects were observed at later time-points. No decrease in cell number was observed when the cells were exposed to 20 mg/ml of 200 nm PPS (Fig. 5 A). Exposure to concentrations of 5?0 mg/ml of CNT decreased cell numbers in a dose-dependent manner at day 7 to approximately 75 ?0 of control cells, respectively. However, with prolonged contact the cell populations recovered. The recovery rate was more rapid for cells exposed to 20 mg/ml than for cells that were exposed to 5 mg/ml and the values reached 90 ?0 of the control after 4 weeks (Fig. 5 B). Cell viability was not impaired at any time-point.Intracellular localization of PPSCells were exposed to 20 mg/ml of the red fluorescent PP.S 3 and 7 at any time-point. However, PPS at 200 mg/ml induced strong activation already after 4 hours, reaching the maximum of an about 50-fold increase after 8 hours and an almost 30-fold increase after 24 hours as compared to non-exposed cells (Fig. 2 A). A similar dose-dependent effect was observed for membrane disruption (Fig. 2 C). Exposure of the cells to 200 nm PPSresulted in a very low release of LDH (up to 15 of the lysis control) and no notable increase in caspase activation at any timepoint (Fig. 2 B and D).Establishment of the microcarrier cell culture systemFor optimization of the exposure system, different coatings of the GEMTM (gelatine, laminin, fibronectin, collagen type I and IV, basal membrane) as well as different incubation protocols, predefined by the supplier, were compared. Cells that were seeded onFigure. 1. Acute cytotoxicity of NPs exerted on EAhy 926 23727046 in different cell cultures after 24 hours. Cells in conventional cultures were treated with NPs dissolved in serum-free medium (A) as well as in medium with 10 FBS (B). Cells cultured on microcarriers were exposed to NMs dissolved in medium with 10 FBS. Data are presented as mean 6 SD. doi:10.1371/journal.pone.0056791.gLong-Term Effects of NanoparticlesFigure. 2. Mode of action of PPS in conventional cultures. Activation of caspases 3 and 7 (A and B) and release of LDH (C and D) upon exposure of EAhy 926 cells to 20 and 200 nm PPS for 4, 8, and 24 hours compared to untreated cells. Data are presented as mean 6 SD. (h), hours. doi:10.1371/journal.pone.0056791.gbasal membrane coated microcarriers and cultured according to the protocol for endothelial cells (HUVEC) reached approximately 4 times higher cell densities compared to those when the protocol for epithelial cells (HEK 293) was used (Fig. 3). Cultures were maintained for 23 days without sub-culturing. The doubling time of EAhy cells was lower in the microcarrier culture system (70 h) than in conventional cultures (30 h). Staining with Hoechst 33342 dye showed an increase in cell proliferation on GEMTM (Fig. 4 A). Furthermore, vital staining for mitochondria and endoplasmic reticulum (ER) demonstrated the viability of the cells (Fig. 4 B).Long-term effects of NPs in microcarrier cultureCells were cultured according to the established protocol (basal membrane coated GEMTM, and incubation protocol for endothelial cells) for four weeks with a medium change performed once per week. After inoculation, NPs were added at a concentration 250 times lower than the concentration where cytotoxicity was seen in the acute cytotoxicity setting (24 hours). Exposure of the cells to 20 mg/ml of 20 nm PPS resulted in a significantly reduced cell number already after 7 days, showing a decrease in cell numbers of approximately 50 . Even stronger effects were observed at later time-points. No decrease in cell number was observed when the cells were exposed to 20 mg/ml of 200 nm PPS (Fig. 5 A). Exposure to concentrations of 5?0 mg/ml of CNT decreased cell numbers in a dose-dependent manner at day 7 to approximately 75 ?0 of control cells, respectively. However, with prolonged contact the cell populations recovered. The recovery rate was more rapid for cells exposed to 20 mg/ml than for cells that were exposed to 5 mg/ml and the values reached 90 ?0 of the control after 4 weeks (Fig. 5 B). Cell viability was not impaired at any time-point.Intracellular localization of PPSCells were exposed to 20 mg/ml of the red fluorescent PP.
