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Preliminary studies of cell culture strategies for bioprocess development based on HEK293 cells


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 [3]; 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).


Characterization of HEK293 cell culture in batch operation was initially performed. It was encountered that cell growth was extended for 168 h reaching approximately 7·106 cell/mL of cell density (Figure 1.1). Nevertheless, maximal cell growth rate (μmax) was only maintained for 96 h. As glucose and lactate were not at limiting concentrations [4], nutrient limitation different from glucose arose as the first hypothesis for this decrease on cell growth rate. Therefore, punctual additions of nutritional supplement for HEK293 were carried out. Xvmax was significantly increased in comparison to basal media, reaching cell densities as high as 17·106 cell/mL (Figure 1.2). Nevertheless, we could not overcome this limit on Xvmax regardless the number of punctual feedings performed. Moreover, nutrient addition did not elongate μmax period (tμ). These results suggested that by-product accumulation different from lactate could be limiting cell growth. In order to validate the hypothesis, complete media replacement (up to three replacements) was studied (Figure 1.3). Although this strategy enabled to extend tμ up to 168 h of cell culture, the maximal cell density reached was similar to nutrient addition strategy (1MR: 12·106 cell/mL; 2MR: 16·106 cell/mL; 3MR: 18·106 cell/mL). This limit on Xvmax encountered on shake flask might be related to a limitation on pO2. Thus, the cell culture system was changed towards a bioreactor with controlled pO2 (maintained between 20-60% of air saturation). In addition, a continuous feeding using a pre-fixed profile addition was implemented. As it can be noticed in Figure 1.4, FedBatch operation in bioreactor enabled to beat the limit encountered in shake flask system, reaching cell densities of 27·106 cell/mL.

Figure 1

Comparison of HEK293 cell growth, viability, glucose and lactate profiles in different cell culture strategies.


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).


  1. 1.

    Nadeau I, Kamen A: Production of adenovirus vector for gene therapy. Biotechnology advances. 2003, 20: 475-489.

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Geisse S, Fux C: Recombinant protein production by transient gene transfer into Mammalian cells. Methods in Enzymology. 2009, 463: 223-238.

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Butler M: Animal cell cultures:recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol. 2005, 68: 283-291.

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Petiot E, Jacob D, Lanthier S, Lohr V, Ansorge S, Kamen A: Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture. BMC Biotechnol. 2011, 11: 84-96.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

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We would like to thank Dr. Amine Kamen (BRI-NRC, Canada) for kindly providing the HEK 293 cell line.

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Correspondence to Leticia Liste-Calleja.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Liste-Calleja, L., López-Repullo, J., Lecina, M. et al. Preliminary studies of cell culture strategies for bioprocess development based on HEK293 cells. BMC Proc 7, P52 (2013).

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  • Shake Flask
  • Transient Gene Expression
  • Media Replacement
  • Viable Cell Density
  • Trypan Blue Exclusion Method