Six well culture dishes

Brain slice cultures of seven-day-old Wistar rats were prepared and maintained as previously described [41 (link)–42 (link)]. Animals were sacrificed and brains were removed and kept under ice-cold conditions. Frontal lobes and Cerebellum were dissected of the hemispheres. The remaining brain was cut into 350 μm thick horizontal slices using a vibratome (Leica VT 1000S, Bensheim, Germany). Brain slices were thereafter transferred onto culture plate insert membrane dishes (Greiner Bio One, Frickenhausen, Germany; pore size 0.4 μm) and subsequently transferred into six-well culture dishes (GreinerBioOne, Frickenhausen, Germany) containing 1.2 ml culture medium (MEM–HBSS, 2:1, 25% normal horse serum, 2% L-glutamine, 2.64 or 14.3 mg/ml glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 μg/ml insulin–transferrin–sodium selenite supplement and 0.8 μg/ml vitamin C). The slices were cultured in humidified atmosphere (35°C, 5% CO₂). The medium was changed on the first day after preparation and from that time forward every other day over a course of 7 days. Invasion fronts were determined by quantifying the tumor infiltrating area.

Organotypic brain slice cultures of 8-day-old C57BL/6 mice were prepared and maintained according to an established protocol [17 (link)]. Animal brains were removed and kept under ice-cold conditions. The cerebellum was removed. The remaining brain was embedded in 5% low melting agarose and cut into 350 µm thick coronal slices using a vibratome (Leica VT1000S, Bensheim, Germany). The brain slices were placed onto cell culture inserts (pore size 0.4 µm; Greiner BioOne, Frickenhausen, Germany) and subsequently transferred into six-well culture dishes (GreinerBioOne) containing 1.2 mL culture medium (MEM-HBSS, 2:1, 25% horse serum, 2% L-glutamine, 2.64 mg/mL glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL insulin-transferrin-sodium selenite supplement, and 0.8 µg/mL vitamin C–pH was adjusted to 7.4 with 37% HCl and the medium was stored at 4 °C for up to 2 weeks). The slices were cultured in a humidified atmosphere (35 °C, 5% CO₂) for up to 21 days. The medium was changed on the day following OBSC preparation and thereafter every second day. At the end of the experiments, the brain slice thickness was reduced to 100 μm, as expected for an organotypic culture.