Figure 3.

Serum-dependence of OSCC cell growth and sphere formation activity.

(A) Serum dependence of OSCC cells. HSC3, HSC4 and SAS cells were cultured in DMEM with various concentrations of serum (0, 1, 2, 5 or 10%; all v/v) for 4–6 days, and cells were enumerated using the MTT assay. The values are means ± SD of data from triplicate samples from one representative experiment. (B) When aggregation culture was performed, 1 × 10³ cells were seeded into each well of low adhesive 96-well plates (Sumitomo, Tokyo, Japan) and cultured in DMEM supplemented with 10% (v/v) FCS at 37 °C under 5% (v/v) CO₂. To allow sphere formation, 1:1000 dilution of a suspension of 1 × 103 cells was added to the well of low-adhesive 96-well U-shaped plates in ‘sphere medium’, which was DMEM/F12 supplemented with 2 mM glutamine, 2% (v/v) B27, 20 ng/ml EGF, 20 ng/ml bFGF, penicillin, and streptomycin. Phase-contrast micrographs of spheres cultured from single SAS cells and single or multiple HSC4 cells growing in low-adhesive 96-well culture plates.