2.4 Induction of target genes
Cells were seeded with 1 mL per well in 24-well plates at a seeding density of 1.0 × 105 cells/mL in serum-containing F12 medium or 1.0 × 106 cells/mL in serum-free medium, and cultured for 4 days. During culture, the medium was changed daily to medium containing E2 (#E2758, Sigma-Aldrich) or 4-OHT at various concentrations. Cell numbers were counted using the trypan blue dye exclusion method.
High-cell-density serum-free culture was performed as follows. After culturing CHO/GEV_scFv-Fc for 3 days in serum-free medium containing 0.1 µM 4-OHT, cells were adjusted to a density of 0.5 or 1.0 × 107 cells/mL. Thereafter, 10 mL of cell suspension was seeded in a bioreactor tube and cultured for 15 days. The culture medium was replaced with fresh medium every day, and the number of cells was counted at that time.
Drug-induced GFP expression in CHO/GEV_GFP was analyzed using a flow cytometer (SH800). scFv-Fc secreted into the culture medium was quantified using an enzyme-linked immunosorbent assay method[30]. The scFv-Fc specific production rate (pg cell-1 day-1) was calculated from scFv-Fc concentrations in the spent medium and numbers of viable cells. Glucose and lactate concentrations were measured using commercially available kits (Glucose Assay Kit-WST, #G264, Dojindo, Kumamoto, Japan; Lactate Assay Kit-WST, #L256, Dojindo).