Culture of Caco2 cells. Acute exposure (24h) for ToF XXXX For ToF-XXXX analysis, cells were cultivated on wafers (polished size up) in 24 xxxxx plates. Caco2 cells were seeded at a density of 20 000 cells/cm². Culture medium was changed three times a week. Cells were differentiated during 25–27 days post-seeding. NPs (NM103 and NM104 provided by the JRC) were dispersed in a mixture of water/BSA 0.05 % (p/v) according to the NANoREG Guidance Document. Differentiated Caco2 were incubated with 3 concentrations of each NP (0.75, 3.13 and 12.5 µg/cm2) (2 replicates per concentration) for 24h. After exposure the cells were rinsed twice with NH4CO3 (144 mM), collected gently, fast-frozen in liquid nitrogen and freeze-dried for the ToF-XXXX analysis. Repeated exposure with or without a recovery period Cells were seeded at 20.000 cells/cm². Culture medium was changed three times a week. Cells were differentiated during 21 days post-seeding. Cells were repeatedly exposed to NM103 or NM104: medium was changed every two or three days and replaced by new medium supplemented with fresh NP solution prepared according to the NANoREG Guidance Document. Cells were exposed for 1 day, 1 week or 2 weeks. In some cases, exposure was followed by a recovery period of up to 1 week as described below. The concentrations were: From 6.5 to 50 µg/cm2 for 24h exposure From 3 to 12.5 µg/cm2 for 1 and 2 weeks exposure. Concentrations tested: - 50 and 12.5 µg/cm2 for 24h recovery - 12.5 and 3 µg/cm2 for longer times Medium changed every 2 or 3 days Treament phase with NM Recovery phase without NM Sampling Treatment schedule for acute and repeated exposure of differentiated Caco2 cells to NM103 and NM104 followed or not by a recovery period (up to 1 week). After exposure, the cells were harvested for estimation of cytotoxicity (Neutral Red Uptake) and IL8 release. Immunostaining of toxicity markers linked with a TEM detection was performed for High Content Screening.
