Abstract
The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of 106.75 and 106.67 TCID50 per 50 µl; signifying a specific productivity of 0.89 and 1.07 TCID50/c.
Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×106c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×108 TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×109 TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition.
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References
Bjare U and Räbb I (1985) Serum free cultivation of lymphoid cells for virus and interferon production. Dev Biol Standard 60, 349–354.
Büntemeyer H (1988) Entwicklung eines Perfusionssystems zur kontinuierlichen Kultivierung tierischer Zellen in Suspension. Ph. D. Thesis, Universität Hannover/D.
Cinatl J Jr, Cinatl J, Rabenau H, Rapp J, Kornhuber B and Doerr H-W (1993) Protein-free culture of Vero cells: A substrate for replication of human pathogenic viruses. Cell Biol Int 17, 885–895.
Croughan MS and Wang DIC (1991) Hydrodynamic effects on animal cells in microcarrier bioreactors. In: Animal Cell Bioreactors, eds. Ho, SC and Wang, DIC, pp. 213–249, Butterworth-Heinemann, Boston, USA.
Duchene M, Peetermans J, D'Hondt E, Harford N, Fabry L and Stephenne J (1990) Production of poliovirus vaccines: past, present, and future. Viral Immunol 3, 243–272.
Enders JF, Weller TH and Robbins FC (1949) Cultivation of Lansing strain of poliomyelitits virus in culture of various human embryonic tissues. Science 109, 85–87.
Fabry L, Baijot B, D'Hondt E and Duchene M (1989) High density microcarrier cell culture for viral vaccine production. In: Advances in Animal Cell Biology and Technology for Bioprocesses, eds. Spier RE, Griffiths JB, Stephenne J and Crooy PJ, pp. 361–365, Butterworths, Sevenoaks/UK.
Furusawa E and Cutting W (1962) Inhibitory effect of ammonium sulfate on Columbia SK virus propagation in mouse ascites tumor cells in vitro. Proc Soc Exptl Biol Med 111, 71–75.
Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM and Bull AT (1991) Glucose-limited chemostat culture of chinese hamster ovary cells producing recombinant human interferon-gamma. Biotechnol Bioeng 39, 327–335.
Hu WS, Meier J and Wang DIC (1985) A mechanistic analysis of the inoculum requirement for the cultivation of mammalian cells on microcarriers. Biotechnol Bioeng 27, 585–595.
Ito Y, Kimura Y, Nagata I and Kunii A (1974) Effects of L-glutamine deprivation of growth of HVJ (Sendai virus) in BHK cells. J Virol 13, 557–566.
Jensen EM and Liu OC (1961) Studies of inhibitory effect of ammonium ions in several virus-tissue culture systems. Proc Soc Exptl Biol Med 107, 834–838.
Merten O-W, Hannoun C, Manuguerra J-C, Ventre F and Petres S (1996) Production of influenza virus in cell cultures for vaccine preparation. In: Novel Strategies in Design and Production of Vaccines, eds. Cohen S and Shafferman A, pp. 141–151. Plenum Press, New York/USA.
Merten O-W, Kierulff KV, Castignolles N and Perrin P (1994) Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: use of various cell lines. Cytotechnology 14, 47–59.
Merten O-W, Petres S and Couvé E (1995) A simple serum-free freezing medium for serum-free cultured cells. Biologicals 23, 185–189.
Montagnon BJ, Fanget B and Nicolas AJ (1981) The large scale cultivation of Vero cells in microcarrier culture for vaccine production preliminary results for killed poliovirus vaccine. Dev. biol. Standard. 47, 55–64.
Montagnon BJ, Vincent-Falquet JC and Fanget B (1984) Thousand litre scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results. Dev Biol Standard 55, 37–42.
Morgeaux S, Tordo N, Gontier C and Perrin P (1993) β-propiolactone treatment impairs the biological activity of residual DNA from BHK-21 cells infected with rabies virus. Vaccine 11, 82–90.
Perrin P, Madhusudana S, Gontier-Jallet C, Petres S, Tordo N and Merten O-W (1995) An experimental rabies vaccine produced with a new BHK-21 suspension culture process: use of serum-free medium and perfusion-reactor system. Vaccine 13, 1244–1250.
Smith BJ and Panico KA (1985) Automated analysis of ophtalaldehyde derivates of amino acids in physiological fluids by reversed HPLC. J Liquid Chromatogr 8, 1783–1795.
Tomei LD and Issel CJ (1975) Growth and Immunogenicity of foot and mouth disease virus in bady hamster kidney cells adapted to and continuously grown in a serum-free chemically defined media. Biotechnol Bioeng 17, 765–778.
van Wezel AL (1967) Growth of cell strains on microcarriers in homogeneous culture. Nature 216, 64–65.
WHO (1988) Expanded programme on immunization. Global eradication of poliomyelitis by the year 2000. Weekly Epidemiological Record 63, 161–162.
WHO (1990) Manual for the virological investigation of poliomyelitis. In: Global Poliomyelitis Eradication by the Year 2000, pp. 37–40.
Zwartouw HT and Algar DJ (1968) Growth of high-titre Semliki Forest Virus in concentrated suspensions of chick embryo cells. J Gen Virol 2, 243–250.
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Merten, OW., Wu, R., Couvé, E. et al. Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors. Cytotechnology 25, 35–44 (1997). https://doi.org/10.1023/A:1007999313566
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DOI: https://doi.org/10.1023/A:1007999313566