Cell density effect in the baculovirus‐insect cells system: A quantitative analysis of energetic metabolism

Abstract

The cell density effect (i.e., the drop in the specific productivity in the baculovirus-insect cells expression system when cells are infected at high cell densities) has been extensively described in the literature. In this article, a model for the central metabolism of serum-free suspension cultures of Spodoptera frugiperda Sf9 cells is proposed and used to investigate the metabolic basis for this phenomenon. The main metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acids cycle, glutaminolysis, and amino acids metabolism), cellular growth and energetics were considered. The analysis of the stoichiometric model allowed further understanding of the interplay of the consumption of carbon and nitrogen sources in insect cells. Moreover, metabolic flux analysis revealed that Sf9 cells undergo a progressive inhibition of central metabolism when grown to high cell densities, for which the incorporation of amino acids carbon backbones into the TCA cycle (mainly glutamine) and the down-regulation of glycolysis are partially responsible. Following infection by baculovirus and cellular division arrest, central energy metabolism depended on the infection strategy chosen (cell concentration at the moment of infection and multiplicity of infection), inhibition being observed at high cell densities. Interestingly, the energetic status of the culture correlated with the decrease in cellular production of baculovirus, meaning that there is room for process optimization through the application of metabolic engineering techniques.

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

“…However, the consumption rate of infected cells at the low ICD was significantly higher than that at the high ICD at the later stage of infection, 24-60hpi (Table 4.3). The consumption rate of infected Sf9 cells in the current study was similar to that reported by Bernal et al (2009), andWong et al (1994), whereas, the glucose consumption rate for uninfected Sf9 cells (25.6×10 -12 mmol/ cell/hour) was lower than most values reported in the literature. The discrepancy in the glucose consumption rate on a per cell basis among the literature may result from the differences in cell size.…”

“…4.7) for the high ICD in comparison with that for the low ICD cases. Glucose consumption rates for uninfected and baculovirus infected Sf9 cells have been extensively reported in the literature (Benslimane et al, 2005;Bernal et al, 2009;Kamen et al, 1996;Radford et al, 1997;Raghunand and Dale, 1999;Rhiel et al, 1997;Wong et al, 1994), but the values vary considerably, possibly due to cell size differences reported for the Sf9 cell line used. The consumption rate varied in the range of 4.52-12×10 -11 and 2.07-24.2×10 -11 mmol/cell/hour for uninfected and infected Sf9 cells propagated in Sf900™II medium, respectively.…”

The components of shear stress affecting insect cells used with the baculovirus expression vector system

“…Thus, the cells are known to produce significant amounts of lactate when suffering oxygen limitation [12]. Here, lactate formation can be detected in both the uninfected and the infected HFM cultures (Figure 6).…”

“…According to this phenomenon, the metabolism of the insect cells is downregulated, when the cells are kept at high density [12,58].…”

“…Depending on the genetic construct, the recombinant protein can accumulate in the cell or can be secreted into the medium. During the infection, the virus takes complete control of the host cell, increasing its catabolic metabolism and thus causing higher oxygen consumption [11,12]. The infected cells are arrested in the G2/M phase [13] and cytoskeleton is destabilized [14,15].…”