Cell line and culture
HEK293SF cells were maintained in serum-free suspension cultures at
37°C, 5% CO2, and 75% relative humidity in a shaker
incubator (Infors, Switzerland) at 135 rpm speed of agitation or
specified otherwise. The basal maintenance and production medium was
HyCell TransFx-H (Cytiva Life Sciences, USA) supplemented with 0.1% w/v
of Kolliphor P188 (Sigma-Aldrich, USA) and 4 mM GlutaMAX (Gibco, USA).
GlutaMAX was replaced by 6 mM L-Glutamine (Gibco, USA) for experiments
in which nutrients and metabolites were analyzed. The supplemented
medium was prepared by adding either glucose for a final concentration
of 7.2 g/L or 15% (v/v) Cell Boost 5 (Cytiva Life Sciences, USA) to
achieve a similar glucose concentration.
Low- and medium-cell-density cultures (1 and 4 × 106cells/mL, respectively, at the time of transfection - TOT) were done in
125-mL polycarbonate shake flasks (TriForest, USA) with a working volume
of 25 mL. High-cell-density (10 × 106 cells/mL at TOT)
transfection was performed in 50-mL TubeSpin bioreactors (TPP,
Switzerland) with a working volume of 10 mL and 180 rpm. For achieving
and maintaining high cell densities, from 48 hours post-seeding, a
perfusion-like mode of operation was followed with medium exchange of 1
vessel volume per day (VVD) both before and after transfection, where
cells were centrifuged and resuspended in fresh medium.
The bioreactor production was performed in a 3-L vessel (Applikon
Biotechnology, The Netherlands) equipped with a double marine impeller,
a pH sensor, a temperature sensor, a dissolved oxygen (DO) concentration
sensor, a micro sparger with 100-nm pore size, and a capacitance probe
for monitoring of cell culture characteristics, such as cell growth. The
pH, temperature, and DO parameters were set at 7.15 ± 0.05, 37°C, and
40% oxygen saturation, respectively. The HEK293SF cells from shake
flasks were inoculated into the bioreactor at 4 × 106cells/mL in a fresh culture medium. The transfection was conducted
immediately after cell inoculation with plasmid delivered on a cell
basis (1 µg/106 cells).