2.3. Kinetic studies in shaken tubes operated in batch and pseudoperfusion (PP) mode
Cells from sorted pools 1 and 2 were seeded at 0.5 × 106 cells/mL into 50-mL spin tubes containing an initial volume of 15 mL HEK TF medium (Xell AG, Germany). Each condition was evaluated in duplicate, and samples were withdrawn daily to monitor cell density and viability, product formation and glucose and lactate concentrations.
Regarding cells grown in pseudoperfusion mode, from day 4 on cells were centrifuged (300 g , 5 minutes), conditioned medium was collected (partially or completely) and fresh medium was added to achieve a culture volume of 10 mL, mimicking an intermittent perfusion process. The proportion of medium exchanged was defined according to the demand, i.e. based on glucose concentration upon sampling: 0.5 vvd (volume of fresh medium per volume of culture per day) when glucose concentration was higher than 2 g/L; 0.75 vvd if between 1 and 2 g/L; and 1 vvd (i.e. complete medium exchange) when glucose was lower than 1 g/L.