HEK-293 cells were derived by Graham in 1977 (Graham et al., 1977
) from human embryonic kidney fibroblasts in which the adenovirus E1A and E1B genes were inserted. HEK-293S cells, adapted to suspension growth, were kindly provided by Dr. Miguel Chillón from the Centre de Biotecnologia Animal i Teràpia Gènica (CBATEG, Barcelona, Spain). HEK-293S cells were maintained in T-flasks (75 cm2
) at 37 °C and 8% CO2
atmosphere and subcultured twice a week at 2–3 × 105
cell/mL. Inocula were prepared in 125, 250, or 500 mL glass spinner flasks (Techne, Burlington, NJ, US) working at 40% of their total volume, stirred at 80 rpm and cells were maintained in the exponential growth phase at a density bellow 10 × 105
cell/mL. Cells were grown in 293 SFM II, a low protein serum-free medium (Invitrogen, Glasgow, UK). The medium was supplemented with 4 mM l
-glutamine prepared in a 200 mM stock solution with 4.5% NaCl (Invitrogen, Glasgow, UK). In bioreactor cultures, 0.1% (w/v) Pluronic F-68 (Sigma-Aldrich, Saint Louis, MO, US) and 50 ppm Antifoam C (Sigma-Aldrich, Saint Louis, MO, US) were also added.
Volume 157, Issue 1, January 2012, Pages 214–222