Culture of ExpiCHO-S and Expi293-F cells

ExpiCHO-S cells (ThermoFisher Scientific Co. ExpiCHO-S Cells, Catalog number: A29127) were suspension-cultured in 25 ml of serum-free ExpiCHO Expression medium in an incubator at 37 °C with a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 125 rpm to a final density of 6 × 10⁶ viable cells/ml. A Gc expression plasmid (15 μg) (pcDNA3.4-TOPO^Gc1F-His, pcDNA3.4-TOPO^Gc1S-His, pcDNA3.4-TOPO^Gc2-His or pcDNA3.4-TOPO^{mouse Gc}) was diluted with 1.0 ml of Opti PRO SFM (ThermoFisher Scientific Co.) and mixed with 80 μl of ExpiFectamine CHO Reagent (ThermoFisher Scientific Co.), previously diluted with 920 μl of Opti PRO SFM. This mixture was incubated for 5 min at room temperature and added to the ExpiCHO-S cell culture flask. The cells were incubated overnight, then 150 μl of ExpiCHO Enhancer and 6 ml of ExpiCHO Feed (ThermoFisher Scientific Co.) were added, and suspension culture was continued for 6–8 days (according to the supplier’s protocol, cell culture can be continued for up to 14 or 15 days). Then, we harvested the cells and media and analyzed the expression levels and sugar modifications of the Gc proteins.

Expi293F cells (ThermoFisher Scientific Co. Expi293F cells, Catalog number: A14528) were suspension-cultured in 30 ml of Expi293 Expression medium in an incubator at 37 °C with a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 125 rpm to a level of 2.5 × 10⁶ cells/ml with > 95% viability. Gc expression plasmids (30 μg) were diluted with 1.5 ml of Opti-MEM (ThermoFisher Scientific Co.) and mixed with 80 μl of Expifectamine 293 reagent (ThermoFisher Scientific Co.), previously diluted with Opti-MEM to 1.5 ml. This mixture was incubated for 20 min at room temperature and then added to an Expi293-F cell culture flask. The cells were incubated under the conditions specified above and, after 18 h, 150 μl of Expifectamine 293 Transfection Enhancer 1 and 1.5 ml of Expifectamine 293 Transfection Enhancer 2 (ThermoFisher Scientific Co.) were added to the flask. The cells and the media were harvested at 3 days post-transfection. The expression levels and sugar modifications of the produced Gc proteins were characterized.