F 12 nut mix glutamax

Neuroblastoma PDX-1, PDX-2, and PDX-3 cells were derived from PDX mouse models established from MYCN-amplified high-risk patient tumors. The PDX cells (also named LU-NB-1, LU-NB-2, and LU-NB-3) have been previously characterized in detail, and cells were cultured as free-floating 3D tumor organoids as previously described [14 (link), 15 (link)]. In brief, cells were cultured in stem cell media [3:1 Dulbecco's Modified Eagle medium and F‐12 Nut Mix GlutaMAX™ supplemented with 2% B27 supplement w/o vitamin A (all Gibco Life Technologies, Carlsbad, CA, USA), 100 IU/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL amphotericin B (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor (PeproTech EC Ltd, London, UK)]. PDX cells were cultured at 37 °C in a humidified atmosphere in 5% CO₂.

Cells were seeded at clonal density of 1000 cells/mL in ultra-low attachment 6-well plates (ThermoFisher^TM Scientific, Fontenay-sous-Bois, France) under neural crest cell culture conditions: DMEM GlutaMAX (0.5X; Gibco^®), F12 NutMix GlutaMAX (0.5X; Gibco^®), B27 supplement (1X; Gibco^®, Waltham, MA, USA), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco^®, Waltham, MA, USA), murine EGF (20 ng/mL; peproTech, Rocky Hill, NJ, USA), murine bFGF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA), and insulin (5 μg/mL; Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France).
Cells were dispersed in low-adherent plates and medium was changed twice a week. Spheroids were allowed to grow over the course of 12 days. Spheres were counted and measured every 2–3 days under the microscope.