Enriched Cancer Stem Cell Culture

Enriched-cancer stem cells (CSCs) were obtained under specific culture conditions as reported in the literature (Brown et al., 2017 (link); Bassi et al., 2020 (link)) as a sarcosphere-forming method starting from a human MG63 osteosarcoma cell line. The MG63 cell line was seeded in Ultra-Low Attachment T25 flasks (Corning Inc., NY) with a density of 2.0 × 10³ cells/cm² in serum-free DMEM F12-GlutaMAX™ Modified Medium supplemented with a specific cocktail of factors: 10 μL/ml N2 (Gibco), 20 μL/ml B27 (Gibco), 0.1 μL/ml human Basic-Fibroblast Growth Factor (bFGF) (Invitrogen), and 0.01 μL/ml human Epidermal Growth Factor (EGF) (PeproTech). The cocktail was added to each flask every 2/3 days for a total of 10 days of culture. After their formation, the CSCs were collected and centrifugated for 10 min at 130 × g; the pellet was resuspended in the same medium conditions, well mixed, and directly seeded in Ultra-Low Attachment 96 well-plate and Ultra-Low Attachment 6 well-plate with 200 µL/well and 1.5 ml/well volume of cell culture medium, respectively. The factors’ cocktail was added every 2/3 days during the experiment following the above-reported manufacturer’s instructions.