2.1. Cell line development, culture conditions and
stability evaluation
In order to generate a cell line capable of stably expressing the
structural premembrane (prM) and envelope (E) proteins, HEK293SF-3F6
cells (NRC, Canada) were transfected by lipofection in a shaken vented
tube (spin tube, TPP AG, Switzerland) using a chemically-defined medium
(HEK TF, Xell AG, Germany) and Lipofectamine 3000 reagent (Invitrogen,
USA), as described previously (Alvim et al., 2019). Transfection volume
was 4 mL.
The gene construct used (named LECC15) was based on an in-house designed
signal peptide followed by the sequence encoding prM-E (683 amino acids)
of the yellow fever virus (South-America I genotype, strain BeH655417,
GenBank # JF912190). The gene construct was codon-optimized,
synthetized, subcloned into pCIneo vector (Promega, USA) and supplied
ready to transfect by Genscript (USA).
Approximately 48h post-transfection (hpt), cells were transferred to a
vented 125 mL-erlenmeyer flask (Corning, USA) and diluted with fresh HEK
TF medium in the presence of 100 μg/mL G418 sulfate (Gibco, USA) for
selection of stable transfectants. Every 3-4 days, viable cell
concentration was adjusted to 1 × 106 cells/mL by
diluting cell suspension with fresh HEK TF medium containing 100 μg/mL
G418 sulfate. Documented research cell banks of the stable cell pool
were cryopreserved at 30 and 60 days post-transfection.
In order to evaluate production profile over time, the cell pool was
kept in culture in the presence of the selection antibiotic for up to
approximately 120 days post-transfection. During the whole period, cells
were kept in a humidified incubator at 37 °C and 5 %
CO2 and shaken at 180 rpm (50 mm orbit). Cell count was
performed using an automated cell counter (Vi-Cell®XR, Beckman Coulter, USA) and, when necessary, glucose and lactate
concentrations were monitored using a YSI 2700 Select Biochemistry
Analyzer (YSI Inc., USA).