Preliminary studies of cell culture strategies for bioprocess development based on HEK293 cells
© Liste-Calleja et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
The use of human embryonic kidney cells (HEK293) for recombinant protein or virus production has gained relevance along the last years. They are specially recommended for transient gene expression and adenovirus or adeno-associated virus generation [1, 2]. To achieve high volumetric productivities towards bioprocess optimization, the concentration of biocatalizer (i.e. animal cells) must be enhanced. The limits for cell growth are mainly related to the accumulation of metabolic by-products, or the depletion of nutrients ; therefore, cell cultures strategies must be developed. In this work, we have explored Punctual Feeding and Media Replacement cell culture strategies to over perform the limit on Xvmax encountered on batch culture mode. Finally, we scaled up cell culture in order to control other parameters (i.e. pO2) that could be limiting cell growth.
Materials and methods
The cell line used in this study was HEK293SF-3F6 (kindly provided by Dr. A.Kamen, NRC-BRI). The basal medium for all cell cultures was SFMTransFx-293 (Hyclone, Thermo Scientific) supplemented with 5% (v/v) of FBS and 4 mM GlutaMAX (Gibco, Invitrogen). For Punctual Feeding and FedBatch Fementation Cell Boost 5 (Hyclone, Thermo Scientific) was used. Batch, media replacement and punctual feeding experiments were performed in 125-ml plastic shake flasks (Corning Inc.) shaken at 110 rpm in an orbital shaker at 37°C, 95% humidity, 5% CO2 incubator. FedBatch Fermentation was carried out in Bioreactor Braun-MCD (2 L) with mechanical agitation at 80 rpm, pH set point 7.1 and pO2 set point 50%. Viable cell density and viability were determined by trypan blue exclusion method and manual counting using a haemocytometer. Glucose and lactate were analysed in an automatic analyser, YSI (Yellow Springs Instrument, 2700 Select).
Punctual feeding and media replacement overcame the limit of 7·106 cell/mL encountered in batch mode operation indicating that nutrient depletion was one of the causes of that limit. Nevertheless, the elongation of tμ found out performing MR suggests that the accumulation of by-products might not be ruled out.
The new limit on Xvmax (≈17-18·106 cell/mL) encountered regardless the cell culture strategy, was outperformed by transferring O2 more efficiently in bioreactor system, reaching cell densities as high as Xvmax = 27·106 cell/mL. The monitoring and control of cell culture parameters (i.e. pO2, pH) will enable to develop more accurate feeding strategies in order to achieve higher cell densities than those presented here (on going work).
We would like to thank Dr. Amine Kamen (BRI-NRC, Canada) for kindly providing the HEK 293 cell line.
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