Tumor cell culture and generation of 3D spheroids

Multicellular spheroids from C6 tumor cells (CLS Cell Lines Service GmbH, Eppelheim, Germany) were generated by seeding cells in 75 cm² culture flasks filled with Dulbecco's modified Eagle's medium (DMEM). Flasks were base-coated with 1% noble agar (Difco; BD Biosciences, Franklin Lakes, NJ, USA) dissolved in medium. Incubation was performed at 37°C in the presence of 5% CO2 with 100% humidity. Spheroids were screened for signs of necrosis using inverted light microscopy. Following 6 days, spheroids with a diameter of 200–300 µm without a necrotic core were selected for implantation.