Neurocult ns a basal

TS543 cells were plated at density 1 × 104 viable cells/cm² and grown as neurospheres with NeuroCult™ NS‐A basal medium supplemented with NeuroCult™ NS‐A proliferation supplement, 20 ng/ml EGF, 10 ng/ml bFGF, and 0.0002% heparin (Stem Cell Technologies). When diameters of neurospheres reached to approximately 100 μm, neurospheres were dissociated to single cells with mechanical force by pipetting 30–50 times.

Seventy-two hours after treatment with TTFields at 37°C or 38.5°C as described above, the cells were seeded at a density of 1x 10³ cells/mL in 500 µL NeuroCult™ NS-A basal serum-free medium supplemented with 20 ng/mL hEGF, 10 ng/mL bFGF and 2 μg/mL heparin (STEMCELL Technologies Cambridge, US) per well in 24-well Sphera Low-Attachment Surface plates (ThermoFisher Scientific, Waltham, MA, USA) for spheroid formation. The spheroids were photographed after 5 days, and cell spheroids were identified according to their typical shape and size and the percentage of spheroids was calculated. To evaluate secondary spheroid formation, equal numbers of surviving cells of the first round of spheroid-formation were reseeded. For quantification of the percentage of spheroid forming cells, the cells were seeded at one cell per well in 96-well plates. Wells with more than one cell were excluded from evaluation.