Glutamine

U-87 MG and F98 cell lines from ATCC were used. The U87 cell line was established from a human glioblastoma. The cells are often polynuclear with very fine membrane extensions. Their mean doubling time is 30.8 ± 2.5 [42 (link)]. The F98 cell line is a murine glioma. The cells are predominantly spindle-shaped and have an in vitro doubling time of approximately 18 h [43 (link)]. Both cell lines were cultured in the same conditions in plastic flasks. The culture medium was composed of DMEM with 4.5% glucose supplemented with 10% fetal bovine serum and 2 mM glutamine (Dutscher, Bernolsheim, France). The cells were maintained at 37 °C in a humid atmosphere with 5% CO ${}_2$ . For the experiments, cells were seeded at a concentration of 1000 cells/well in 96-well flat CleaLine^® plates for 2D monolayer cultures or in 96-well CELLSTAR^® round-bottom ultra-low attachment plates for 3D spheroid cultures.

The MC3T3-E1 cells were plated in expansion medium composed of alpha-Modified Eagle Medium (α-MEM) (Dutscher) containing 10% FCS, 1% penicillin/streptomycin and 1% glutamine (Dutscher). Osteogenesis differentiation was started when MC3T3 had reached confluence (day 0) by incubation with osteogenic inductors (50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich Corporation) for 14 days.