3.2.1 Cell growth kinetics
The growth kinetics of HEK293T cell cultures in 24-DSW plates and 2 L STR are depicted in Figure 4. The fold change in viable cell density for both vessel systems were comparable for the entire duration of culture (Figure 4A). The first two days correspond to the growth phase where cells proliferate before being induced to initiate LVV production. Within 48 hours of culture, cells in both reactor formats reached the target induction cell density. The decrease in cell density at day 2 corresponds to the induction time point; here, the cell density decreased due to dilution of the culture upon addition of induction agents. After induction, the fold change in viable cell density remained comparable, with both cultures achieving a similar cell density at the harvest time point.
High cell viability values (i.e. > 90 %) were also maintained in both systems throughout the entirety of the culture (Figure 4B). A slight decrease in cell viability in generally observed after 24 hours post induction (i.e. day 3). Studies have linked this with the onset of viral release (Ansorge, Lanthier, Transfiguracion, Henry, & Kamen, 2011; Petiot, Ansorge, Rosa-Calatrava, & Kamen, 2017). As the LVV buds off from the plasma membrane of the producer cell, the integrity of the membrane is compromised which may lead to apoptosis and a decrease in cell viability. A similar trend was also observed in Figure 4B, however, it should be noted that the decrease in viability after day 3 was very marginal with values still above 90% at the end of the culture.
Similar glucose and lactate metabolite concentrations (Figure 4C and 4D respectively) were achieved in both platforms up until the induction time point (after day 2). Thereafter, glucose depletion and resulting lactate accumulation was greater in the 2 L STR compared to the microwell plate. This trend in metabolites post-induction could be explained by the contrasting pH profiles for both systems. Liste-Callejaet al ., 2015 have demonstrated that culture pH below 6.8 triggers lactate co-metabolism in HEK293T cells (Liste-Calleja et al., 2015). This cellular adaptation is used as a pH detoxification strategy by means of co-transporting extracellular protons together with lactate into the cytosol. For the STR, the pH was maintained above 6.8 through the controlled addition of sodium carbonate base and CO2. However, no such control was available in the microwell system which was evident by the significant fluctuations in pH as depicted in Figure 5. From the graph, it can be seen that the microwell pH decreased immediately after induction (i.e. below 6.8) as the cellular metabolic activity increased. Around the same time point, a plateau in lactate accumulation for the 24-DSW plate was also observed (Figure 4D) possibly due to lactate co-metabolism as suggested by Liste-Calleja et al ., 2015. However, despite the disparity in pH control between the two platforms, the 24-DSW plate was still a good scale-down model for the 2 L STR with respect to cell growth and LVV productivity.