Murine Hippocampal Cell Culture Protocol

Primary hippocampal cells were obtained from murine embryos (day 18 of gestation). A detailed protocol for culture preparation is described in Vedunova et al., 2015 (link). Hippocampi were surgically isolated. Cell dissociation was achieved through mechanical dissection followed by incubation for 20 min in 0.25% trypsin-EDTA solution (Gibco, 25200056, United States). The obtained cell suspension was centrifuged at 1000 rpm for 3 min. Then, the cell pellet was resuspended in Neurobasal^TM medium (Gibco, 21103049, United States) supplemented with 2% B27 (Gibco, 175040446, United States), 0.5 mM L-glutamine (Gibco, 25030024, United States) and 5% fetal bovine serum (FBS) (PanEco, K055, Russia). To perform a viability assessment, immunocytochemical analysis and registration of functional calcium activity, we placed cells on coverslips (18x18 mm) pretreated with polyethyleneimine solution (1 mg/mL) (Sigma-Aldrich, Germany). For electrophysiological experiments, cells were cultured on multielectrode arrays (MEAs; MEA60, Multichannel, Germany). The initial density of cells was 9000 cells/mm². Half of the medium containing 0.4% FBS was replaced every third day. Cell viability was maintained under constant conditions of 35.5°C, 5% CO₂ and a humidified atmosphere in a CO₂ incubator (Sheldon Manufacturing, United States).