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.