C chamber hypoxia chamber

U87 and U87 EGFRvIII mutant cells (U87^vIII) were provided by Dr. Nathan Price (Institute for Systems Biology, Seattle, WA). Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin/streptomycin (Lonza) at 37°C in a 5% CO₂ environment and passaged upon confluence. At the start of each experiment, cells were homogeneously mixed with the pre-polymerized hydrogel solution at a density of 1 × 10⁵ cells/25 μl hydrogel then photopolymerized as described above. Cell-seeded hydrogels were incubated in cell culture medium at 37°C, 5% CO₂ in low adhesion well-plates containing standard culture media with ~20% O₂ (normoxia) or 1% O₂ (hypoxia) in a dedicated, computer controlled hypoxia chamber (C-Chamber Hypoxia Chamber, Biospherix^™, Parish, NY) within the cell culture incubator. Media were changed at days 3 and 5 for cultures extending to 7 days. Media exchange for samples in the hypoxia chamber was performed using culture media pre-conditioned in the hypoxia chamber for at least 12 hours.