Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Lymphoprep medium (Axis Shield) [36] of freshly withdrawn ACD blood from a healthy donor. They were seeded in a 96-well plate (200,000 cells/well) in 200 µL complete culture medium (RPMI, 10% FCS), supplemented or not with the polyclonal activator phytohaemoagglutinin (PHA, 5 mg/mL) in presence of 0, 1, 25, 50, 100 µg NPs/mL in culture medium. Such NPs concentrations were obtained by dilution of a 10 mg NPs/mL stock suspension pre-filtered trough 22 µm nylon mesh filter (Millipore, Darmstadt, Germany). PHA is a polyclonal activator promoting agglutination by close contacts between cell membranes, hence stimulating cell division and metabolism, used as positive control for stimulation of (otherwise resting) human PBMC and induction of functional T-lymphocytes in vitro.

Under these conditions, lympho/monocytes (either unstimulated or PHA-activated), were used as target cells in vitro to evaluate the cyto/immunotoxicity after being induced by exposure for 6, 24, 48, 72 h in humified 5% CO2, 37 °C by TiO2@MSN, MSN and nano-TiO2 (Figure 1).

Study outline.

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