Effect of the peak cell density of recombinant AcMNPV-infected Hi5 cells on baculovirus yields

Abstract

The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and β-galactosidase (β-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the β-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.5 and 6 × 10(6) cells/mL and a multiplicity of infection of 10 plaque-forming units (PFU)/cell either in the original or fresh medium conditions. There were negative correlations between the three key virus infection indicators (vDNA, mRNA and β-Gal) and the peak cell density (PCD). However, unlike infected Sf9 cells, the yield decline started at the lowest PCD investigated (0.6 × 10(6) cells/mL). Generally, the yield decline with increasing PCD was most pronounced for β-Gal followed by mRNA and was more moderate for vDNA. The decline was significantly reduced but not totally arrested when fresh medium replacement was used. The results suggest that the reduction in recombinant protein-specific yields at high PCDs is associated with limitations during the up-stream processes of replication and transcription rather than entirely caused by limitations during translation. In addition, low production rates at late infection stages of moderate to high ICDs are a probable cause of the cell density effect.

Integrating nanoparticle quantification and statistical design of experiments for efficient HIV-1 virus-like particle production in High Five cells

“…BV production was also significantly reduced in such conditions. These differences in production could be related to the cell density effect previously described in insect cells with the BEVS Huynh et al 2013;Huynh et al 2014). On the other side, medium replacement before BV infection moderately increased VLP production but duplicated baculovirus production.…”

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution

“…High Five cells were efficiently adapted to grow at neutral culture pH (7.0), showing similar specific growth rates (≈ 0.04 h −1 ) and morphologic characteristics (round shape with diameter ≈ 16-17 µm [31,32]) to non-adapted cells. In addition, adapted cells were efficiently maintained in culture for over 100 population doublings, thus demonstrating their stability to be routinely cultured in such a non-standard condition.…”

Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production

“…Centrifuging cells at 2 × 10 6 cells/mL to concentrate before transfection proved to work, suggesting that the referred CDE might be related and conditioned by the intrinsic metabolic state of the cells. The same problem appears when working with insect cell lines …”

“…The same problem appears when working with insect cell lines. 29 With the implementation of perfusion in bioprocessing productions, cell cultures are able to reach very high densities up to 50−150 × 10 6 cells/mL. 30,31 The rapid advances in the field are making essential the need to characterize metabolic bottlenecks regarding transfection and production and provide some insights into the metabolic causes of the CDE.…”