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Improving Influenza virus production in HEK293 cell culture supplemented with animal-free insulin

Improving Influenza virus production in HEK293 cell culture supplemented with animal-free insulin

Authors: Aziza P. Manceur, Sonia Tremblay, Sven Ansorge

Background

Cell-culture based vaccines are a valuable alternative to egg-produced vaccines. HEK293SF-3F6 is a suspension cell line used to produce Influenza virus, in flasks and large bioreactors. HEK293SF-3F6 cells grow in commercially-available serum-free media but show weak growth profiles. Insulin has been proved to positively affect cell survival, by promoting anti-apoptotic and mitogenic pathways, and to interact with cellular signalling pathways exploited by the influenza virus.
This study addresses the effect of recombinant insulin supplementation on HEK293SF-3F6 cell growth and Influenza virus production.

Study Description

Different doses of insulin were supplemented to HEK293SF-3F6 cells grown in chemically-defined serum-free media and viable cell density was measured. Influenza virus production was measured as HA concentration, following HEK293SF-3F6 transduction with Influenza virus. Phosphorylated Akt was measured as indication of Akt pathway activation in response to insulin addition.

Results

Addition of 10-20mg/L of insulin every 72hrs resulted in up to 4-fold increase of viable cell density of HEK293SF-3F6 cells (fig. 1a). Addition of 25-100 mg/L insulin at time of infection increases the yield of influenza (H1N1 A/Puerto Rico 8/34) virus by almost 2-fold (fig. 1b, left panel), without affecting the total cell count at harvest (fig. 1b, right panel). Addition of insulin to HEK293SF-3F6 cells transfected with influenza virus showed increased Akt activity (fig. 1c).

 

Insulin increases influenza virus yield in HEK293 cells for vaccine production

 

Insulin increases influenza virus yield in HEK293 cells for vaccine production 1

 

Insulin increases influenza virus yield in HEK293 cells for vaccine production 2

Figure 1: Effects of insulin supplementation to HEK293SF-3F6 cells. Viable cell density following supplementation of 10 and 20 mg/L insulin (A). Concentration of influena virus (left panel) and total cell number (right panel) at different insulin concentrations 5-200 mg/L (B). Akt acitivity measured in response to Influenza virus transfection with or without insulin supplementation (C).

Conclusion

  • Growth limitations observed in a chemically-defined media are alleviated by insulin.
  • Insulin increases the yield of influenza virus in a 24-well microbioreactor.
  • Insulin is a strong activator of the PI3K/Akt pathway, which plays a key role in influenza production.

Further information

In case of any questions, please contact our Global Product Manager Sara Bursomanno at usbu@novonordisk.com

Read and download the case study here. You can download the scientific poster based on the study here.

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Email: chju@novonordisk.com