3.4 Suspension culture of CHO/GEV_scFv-Fc cells in serum-free medium
For production of useful substances using CHO cells, suspension culture with serum-free medium is widely employed. Due to the absence of serum components, the responsiveness of cell proliferation and exogenous gene expression to the inducing agent 4-OHT may vary. As the drug response may become more sensitive compared with conditions using serum-containing medium, we investigated the impact of 4-OHT concentration on scFv-Fc production for CHO/GEV_scFv-Fc cells cultured in a commonly used serum-free medium (Figure 4). CHO/GEV_scFv-Fc cells were seeded at a concentration of 1.0 × 106 cells/mL in 2 mL of serum-free medium containing various concentrations of 4-OHT and cultured in a 24-well cell culture plate. The medium was changed daily to fresh medium containing 4-OHT, and cells were cultured for 4 days. Cell proliferation and viability are shown in Figure 4A and 4B, respectively. Only a slight decrease in cell proliferation was observed at 4-OHT concentrations below 0.1 µM, and the viability remained close to 100% throughout the culture period. In contrast, significant decreases in cell proliferation were observed, particularly at 0.5 µM and 1.0 µM. Additionally, cell viability was affected with the 1.0 µM addition condition, dropping to around 80% by day 4 of culture. To evaluate scFv-Fc production over the culture period, we increased the seeding cell density 10-fold compared with the serum-containing culture. Under this condition, the scFv-Fc concentration reached 42 µg/mL (Figure 4C), which is 8.6-fold higher than that of serum-containing culture. However, specific productivity was decreased in conditions with 0.5 µM and 1.0 µM 4-OHT, which showed good values in serum-containing cultures. With 0.1 µM 4-OHT, the maximum specific productivity was 13 pg cell–1 day–1 (Figure 4D), comparable to the production level observed in serum-containing cultures with the same 4-OHT concentration (15 pg cell–1day–1). These results confirm that drug-inducible transgene expression can be achieved even with serum-free medium, and the optimal concentration of 4-OHT is approximately 10-fold lower compared with serum-containing culture.