study of r-insulin supplementation in expisf9 cell … · 2020. 5. 15. · universitat autònoma de...
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STUDY OF r-INSULIN SUPPLEMENTATION IN EXPISF9 CELL CULTURES FOR CELL GROWTH AND BACULOVIRUS-DRIVEN HIV-1 VLP PRODUCTION
Díaz-Maneh, A., Puente-Massaguer, E., Cervera, L., Gòdia, F. Cell and Bioprocess Engineering Group
Departament d’Enginyeria Química, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain [email protected]
The aim of this study was to investigate the effect of r-insulin in Sf9 cell cultures growing in SF CD medium. An initial phase of adaptation to different r-insulin concentration was performed prior to
ACKNOWLEDGEMENTS
The insulin used in this work was kindly provided by Novo Nordisk Pharmatech A/S (Koege, Denmark). Fruitful discussions with Sara Gualdoni and Vanessa León are acknowledged.
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Cell growth kinetics of Sf9 cells in Sf9 CD medium.A. Viable cell concentrations and viabilities of Sf9 cells at different r-insulin concentrations (n=3). B. Comparison of viable cell concentrations and viabilities of Sf9 cells with 1 mg/L and withoutr-insulin supplementation (n=3).
r-insulin adaptationAdaptation phase to different r-insulin concentrations (1, 3, 5 mg/L). NI: No insulin.
Gag-eGFP VLP concentrations measured using Nanoparticle Tracking Analysis (NTA) A. VLP concentration. B. Size distribution of fluorescent particles with 1 mg/L supplementation.
Gag-eGFP production kinetics in supernatant at different r-insulin concentrations The results of triplicate experiments are shown.
B A
REFERENCES
GA
G
Baculovirus preparation
Kinetics of infection
Cell density and viability
VLP quantitation
Gag-eGFP quantification
Cytometry
Cell counter
Nanoparticle tracking
analysis (NTA)
Fluorometry
sf9 cells
Harvest at 72 hpi
n = 3
Cell growth Sample every 24h
Cervera, L. et al. Journal of Biotechnology.166, 152–165 (2013). Puente-Massaguer, E. et al. Engineering in Life Sciences. 1–13 (2019).
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24hpi 48hpi 72hpi
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Viability NI Viability 1 mg/L Viability 3 mg/L Viability 5 mg/L
dt1/2 (NI) = 23.8 ± 2.4 h dt1/2 (1) = 21.0 ± 0.5 h dt1/2 (3) = 22.1 ± 0.5 h dt1/2 (5) = 21.2 ± 0.5 h
Growth kinetics of Sf9 during BV infection at MOI = 1 Viable cell concentrations and viabilities of triplicate experiments are shown.
BV-Gag-eGFP infection kinetics Gag-eGFP baculovirus infection kinetics of Sf9 cells. Triplicate experiments are depicted.
NI 3 mg/L
1 mg/L 5 mg/L
r-INSULIN SUPPLEMENTATION OF SF9 CELLS
r-INSULIN SUPPLEMENTATION IN BV-INFECTED SF9 CELLS
r-INSULIN ADAPTATION PHASEMETHODOLOGY
OUTLINE
BV Infection
n = 3
1.2-fold A B
100 – 200 nm
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CONCLUSIONS
A 1.2-fold improvement in maximal viable cell concentration was obtained with 1 mg/L r-insulin
dt1/2 (NI) = 23.8 ± 2.4 h dt1/2 (1) = 21.0 ± 0.5 h
A 1.2-fold increase in VLP production was attanined with 1 mg/L r-insulin A 1.1-fold reduction in dt1/2 was achieved with 1 mg/L r-insulin supplementation
experimentation. The first part focused on the evaluation of r-insulin as a supplement for cell growth and viability maintenance. In the second part, r-insulin was investigated as an enhancer for HIV-1 Gag VLP production through baculovirus infection (BV). The Gag gene was fused in frame to eGFP to ease process characterization and product quantification.
sf9 cells