Astrocytes

Complex primary rat cultures containing cortical neurons (Lonza, Walkersville, MD, United States), astrocytes (Lonza), and oligodendrocyte precursor cells (ScienCell, Carlsbad, CA, United States) were prepared as previously described (Enright et al., 2020 (link)). Briefly, 6-well Multi-channel system MEA devices (Multichannel Systems, Reutlingen, Germany) or wells in a 96-flat bottom-well plate (Corning) were coated with 0.1 mg/ml poly-D-lysine (Millipore-Sigma) and washed with sterile DI water (4X). Cells were seeded at a density of 3,031 cells/mm² on devices and plates with a seeding percentage of 79% neurons, 16% astrocytes and 5% oligodendrocyte precursor cells, based on previous reports of the brain cells’ ratio during postnatal periods (Bandeira et al., 2009 (link); Clarke and Barres, 2013 (link)) and our previous work (Enright et al., 2020 (link)). Cultures were maintained in PNGM™ Primary neuron growth medium bulletkit™ (Lonza) in a humidified incubator (37°C, 5% CO₂). For MEA devices, custom device caps, made from polytetrafluoroethylene (PTFE) housing and a fluorinated ethylene-propylene (FEP) membrane (ALA Scientific, Farmingdale, NY), were used to maintain sterility and to allow for gas exchange. Cultures were fed every 3–4 days with 50% media exchange.

Primary normal human astrocytes were obtained from Lonza. Cells were cultured in astrocytes medium supplemented with 0.1% rhEGF, 0.25% Insulin, 0.1% Ascorbic Acid, 0.1% GA-1000, 1% L-Glutamine, and 3% FBS (all supplements from Lonza, Germany).

All cells were maintained at 37°C and 5% CO₂. C57BL/6N cortical neurons, striatal neurons, and astrocytes were purchased from Lonza (Basel, Switzerland), and microglia were purchased from ScienCell (Carlsbad, CA). Neurons were plated at approximately 1.04 × 10⁵ cells/cm² in vendor-recommended medium, and a medium exchange was performed after 2 hours. astrocytes were plated at approximately 1.32 × 10⁴ cells/cm² in vendor-recommended medium, and a medium exchange was performed after 6 hours. microglia were plated at approximately 2.19 × 10⁴ cells/cm² in vendor-recommended medium, and a media exchange was performed after 24 hours. After 3 days in culture, cells were treated with 10 μM EFV, 8-OHEFV, 8,14-diOHEFV, or 0.1% DMSO vehicle control for approximately 24 hours. Cells were pelleted, and culture media was removed, dried down, and resuspended in 100% methanol. Metabolite formation was detected using a Q Exactive Orbitrap as described above.