HEK293 cell culture media study: increasing cell density for different bioprocess applications
© Liste-Calleja et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among them, media development is of major interest . According to the increasing constraints regarding the use of animal derived components on industrial bioprocesses but also the drawbacks of its depletion from cell culture , the main goal of the present work was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained.
Materials and methods
The cell line HEK293SF-3F6 employed in this study was kindly provided by Dr. A.Kamen, NRC-BRI. The basal media tested were CDM4HEK293, SFM4HEK293 and SFMTransFx-293 (Hyclone, Thermo Scientific) supplemented -when indicated- with FBS (Invitrogen) and/or Cell Boost 5 (80 g/L) (Hyclone, Thermo Scientific). Viable cell density and viability were determined by trypan blue exclusion method and manual counting using an haemocytometer. The adenovirus strain HAdV-5(ΔE1/E3) encoding pCMV-GFP was used for infection experiments. All infections were carried out at MOI≈1 TOI≈0.5 × 106cell/mL in 6-well-plate. Harvesting was performed 48 hpi.
Viral titration was performed by Flow cytometry on a FACS Canto (Becton and Dickinson, Bioscience) by adaption of a protocol previously described .
Kinetic parameters for HEK293 cell cultures corresponding to the profiles depicted in Figure 1.
Xvmax (×106 cell·mL-1)
0.85 ± 0.0
3.53 ± 0.21
2.1 ± 0.12
μmax (×10-2 h-1)
1.06 ± 0.01
2.46 ± 0.14
2.43 ± 0.03
Xvmax (×106 cell·mL-1)
6 ± 0.0
4.67 ± 0.48
7.02 ± 0.06
2.61 ± 0.04
2.8 ± 0.05
2.67 ± 0.01
Xvmax (×106 cell·mL-1)
4.11 ± 0.33
7.29 ± 0.18
9.75 ± 0.25
2.1 ± 0.06
2.06 ± 0.03
2.17 ± 0.03
From the range of applications in which HEK293 can be used, the work carried out in this work was directed to recombinant adenovirus production. Hence, the evaluation of the effect of supplementation in the cell media selected on adenovirus infection efficiency and final titer obtained was evaluated (Figure 1C). Efficiency of infection was around 63% as expected for an effective infection  in all conditions. In regards to adenovirus production, FBS increased it up to fivefold, whereas CB5 supplementation did not affect significantly, and the addition of both supplements almost doubled the viral production in comparison to basal medium. It is proposed that an increment of osmolarity due to the addition of both supplements might explain the slight reduction on productivity in comparison to the addition of FBS solely .
Two culture platforms are proposed for two possible scenarios in basis of the Xvmax reached: (1) HyQSFMTransFx-293 CB5 supplemented -10% v/v- for animal derived component Free required bioprocesses (Xvmax= 12.6 × 106 cell/mL) and (2) HyQSFMTransFx-293 FBS and CB5 supplemented -5% and 10% v/v respectively- for animal derived component containing bioprocesses (Xvmax= 16.7 × 106 cell/mL). In both cases, μmax and tμ values were preserved or even improved with respect to basal media and any of the supplements negatively affected the adenovirus production when compared to non-supplemented infections.
We would like to thank Dr. Amine Kamen (BRI-NRC, Canada) for kindly providing the HEK 293 cell line.
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