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.