Dmem high glucose medium

Neonatal astrocytes were prepared from brains of newborn C57BL6 wild type mice according to published protocols [25 (link), 26 (link)]. Briefly, primary mixed glial cultures were established from the forebrains of 1 day-old male and/or female pups, mechanically dissociated and digested with 0.25% trypsin solution. Mixed cultures were maintained in complete astrocyte medium (DMEM-high glucose medium containing 1% antibiotics (Pen/Strep solution), 200 mM l-Glutamine, 100 mM Sodium Pyruvate and 10% FCS (all from Euroclone, Italy)). Ten day-old primary cultures were vigorously shaken to discard microglia and oligodendrocytes. The remaining adherent cells were detached and, following an adhesion step, non adherent cells were reseeded on poly-d-lysine (Sigma) coated flasks. Adult astrocyte cultures were established from 2 months-old male and/or female C57BL6 wild type mice. Brains were extracted and dissociated by combining enzymatic and mechanical dissociation using Adult Brain Dissociation Kit and the gentleMACS™ Dissociator (both from Miltenyi Biotec). After dissociation, myelin, cell debris and erythrocytes were removed and remaining cells were seeded on poly-d-lysine coated flasks in complete medium. Purity of astrocyte cultures was > 95% according to GFAP immunofluorescence.

The PANC-1 and MIAPaCa-2 human pancreatic ductal adenocarcinoma cell lines purchased from Sigma Aldrich (Milan, Italy) were cultured in DMEM high glucose medium (EuroClone, Milan, Italy) supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg streptomycin, and 1 mM sodium pyruvate. Epithelial cell lines derived from the normal human colon (NCM460D) and normal human fibroblast (NHFA12) were used as normal cells for the cytotoxicity assay. NCM460D was cultured in RPMI1640 supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 mg streptomycin. NHFA12 was cultured for the PANC-1 cell line. Cell lines were maintained at 37 °C with 5% CO₂ and 95% humidity.

The ICP analyses summarized in Table 1, except for STe5, were previously published and reported [33 (link),35 (link)]. The glass samples of STe5 were subjected to in vitro bioactivity tests by soaking them in simulated body fluid (SBF). The SBF was prepared using the protocol developed by Kokubo et al. [75 (link)]. Polished glass discs were immersed in 50 mL of SBF for fixed periods (1, 3, 7, 14, 28 days, here reported only 3-day timepoint) with five replicate samples of each glass per time point. Samples were maintained at 37 °C in an incubating shaker with an orbital speed of 120 rpm to simulate the physiological fluid flow. Solution at each time point was collected and the cumulative ion release for each sample was calculated by adding the ion release value at the selected time point to the previous ones. In the case of Te-ion release in cell medium (DMEM high glucose medium (Euroclone, Pero, Italy), supplemented additionally by penicillin/streptomycin and L-Glutamine (1% of both), STe5 specimens were soaked in the medium without FBS, 1.5 mL per disk in triplicates. Samples were maintained in the solution for 3 days, at 37 °C, 5% CO₂ incubation. From both SBF and cell medium collected samples, the Te-ion release was determined via an inductively coupled plasma mass spectrometer (ICP-MS, iCAPTM Q, Thermo Fisher Scientific, Waltham, MA, USA).