2.3. Primary Rat Glial Cell Culture

The primary culture of rat glial cells, used here as the control non-modified normal cells, was isolated from one-day-old Wistar rat pups as described by [28] with the following modifications. The forebrains were isolated, washed with ice-cold DMEM GlutaMAX-1 supplemented with 1% penicillin/streptomycin, and transferred to 0.25% trypsin-EDTA (Gibco 25200056) for 25 min at 37 °C with 5% CO₂. Next, DMEM, supplemented with 10% FBS and antibiotics, was added to the tissue suspension, followed by mechanical dissociation by pipetting, and the suspension was passed through a 70 µm cell strainer (Falcon^® 352350, Glendale, AZ, USA). The single cells were plated into poly-L-lysine-coated (100 µg/mL; Sigma-Aldrich P4707) flasks and maintained in complete DMEM. After 3 days, 2/3 of the medium was refreshed. On Day 7, the flasks with primary glial cultures were transferred to an orbital shaker at 100 rpm for 30 min to remove microglia. After that, the entire medium with detached cells was removed. The homogeneity of the cell population was maintained for at least four passages. More than 80% of the cells were astrocytes as confirmed by immunofluorescence staining for glial fibrillary acidic protein (GFAP). Afterwards, cells were seeded and cultured under experimental conditions.