Study of Vero Cell Growth in A Modified Serum-Free Medium (SFM01-M)

Main Article Content

Manoch Posung, et al


The aim of this work was to study a modified serum-free medium (SFM01-M) in supporting Vero cell growth in both static and microcarrier cultures. SFM01-M was developed using a systematic design of experiments (DoE). In static cultures, a concentration of rhEGF in SFM01-M was first studied in order to find its concentration suitable for using in the long-term subcultures of Vero cells and in an attempt to assess the minimizing of a cost of SFM01-M as a result of the addition of rhEGF. The results shown here indicated that the addition of rhEGF at least 0.015 mg/L in SFM01-M had effect on cell yields, particularly during subcultures under a serum-free condition (P1-P3). Additionally, a comparison of cell yields obtained from P3 between the rhEGF-added SFM01-M and the rhEGF-free SFM01-M cultures was found to be significantly different (P-value < 0.05). Therefore, in the long-term subcultures of Vero cells, a concentration of rhEGF at 0.015 mg/L was chosen to add to SFM01-M. The results showed that the cell yields obtained during P1-P9 were in the range of 7.09±0.12–7.33±0.33x105 cells/well (6-well plate) and then decreased gradually from P10 to P12. With the gradual decrease of growth ability, the cell yields obtained during P12-P20 were rather lower (5.75±0.39–5.96±0.36x105 cells/well) than those obtained from previous passages. By increasing an initial cell density of 4x104 cells/cm2, therefore, the cell yields could be increased in the range of 7.54±0.19–8.00±0.15x105 cells/well during P10-P20. In microcarrier cultures, the ability of Vero cells to attach and grow on CytodexTM1 solid microcarriers in SFM01-M in a spinner flask was found to be close to that of VP-SFM. From this study, Vero cells can be grown in SFM01-M in both static and microcarrier cultures. Moreover, a cost of SFM01-M is relatively cheap (approximately 70 US dollar per liter) and SFM01-M can also be an economical alternative for Vero cell cultures under a serum-free condition.

Article Details



Arigony, A. L. A., De Oliveira, I. M., Machado, M., Bordin, D. L., Bergter, L., Prá, D. & Henriques, J. A. P. (2013). The influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability. BioMed. Res. Int., 2013, 1-22.

Arrigoni, O. & De Tullio, M. (2000). The role of ascorbic acid in cell metabolism: between gene-directed functions and unpredictable chemical reactions. J. Plant Physiol., 157, 481-488.

Babcock, J., Wilcox, C. & Huttinga, H. (2010). Partial Replacement of Chemically Defined Media with Plant-Derived Protein Hydrolysates. Biopharm Int., 23, 36-41

Bettger, W. J., Boyce, S. T., Walthall, B. J. & Ham, R. G. (1981). Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone. Proc. Natl. Acad. Sci. USA., 78, 5588-5592.

Bjelakovic, G., Pavlovic, D., Jevtovic, T., Stojanovic, I. & Sokolovic, D. (2006). Vitamin B12 and Folic Acid Effects on Polyamine Metabolism in Rat Liver. Pteridines, 17, 90-94.

Brunner, D., Frank, J., Appl, H., Schöffl, H., Pfaller, W. & Gstraunthaler, G. (2010). Serum-free cell culture: The Serum-free Media Interactive Online Database. ALTEX, 27, 53-62.

Castle, P. & Robertson, J. S. (1998). Animal Sera, Animal Sera Derivatives and Substitutes Used in the Manufacture of Pharmaceuticals. Biologicals, 26, 365-368.

Cervera, L., Gutiérrez, S., Gòdia, F. & Segura, M. M. (2011). Optimization of HEK 293 cell growth by addition of non-animal derived components using design of experiments. BMC Proceedings, 5, 126.

Chen, Z., Ke, Y. & Chen, Y. (1993). A serum-free medium for hybridoma cell culture. Cytotechnology, 11, 169-174.

Chuman, L., Fine, L. G., Cohen, A. H. & Saier, M. H. Jr (1982). Continuous Growth of Proximal Tubular Kidney Epithelial Cells in Hormone-Supplemented Serum-Free Medium. J. Cell Biol., 94, 506-510.

Chun, B. H., Kim, J. H., Lee, H. J. & Chung, N. (2007). Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture. BioresourTechnol., 98, 1000-1005.

Clark, J. M., Gebb, C. & Hirtenstein, M. D. (1982). Serum supplementations and serum-free media: applicability for microcarrier culture of animal cells. Dev. Biol. Stand., 50, 81-91.

Desai, N. & Goldfarb, J. (1998). Co-cultured human embryos may be subjected to widely different microenvironments: pattern of growth factor/cytokine release by Vero cells during the co-culture interval. Hum. Reprod., 13, 1600-1605.

Eto, N., Yamada, K., Shito, T., Shirahata, S. & Murakami, H. (1991). Development of a Protein-Free Medium with Ferric Citrate Substituting Transferrin for the Cultivation of Mouse-Mouse Hybridomas. Agri. Biol. Chem., 55, 863-865.

Franĕk, F., Hohenwarter, O. & Katinger, H. (2000). Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures. Biotechnol. Prog., 16, 688-692.

Gospodarowicz, D. & Moran, J. S. (1976). Growth factors in mammalian cell culture. Annu. Rev. Biochem., 45, 531-558.

Hassell, T., Gleaves, S. & Butler, M. (1991). Growth inhibition in animal cell culture, the effect of lactate and ammonia. Appl. Biochem. Biotechnol., 30, 29-41.

Heidemann, R., Zhang, C., Qi, H., Rule, J. L., Rozales, C., Park, S., Chuppa, S., Ray, M., Michaels, J., Konstantin, K. & Naveh, D. (2000). The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology, 32, 157-167.

Hewlett, G. (1991). Strategies for optimising serum-free media. Cytotechnology, 5, 3-14.

Jayme, D. W. (1991). Nutrient optimization for high density biological production applications. Cytotechnology, 5, 15-30.

Jan, D C H., Jones, S. J., Emery, A. N. & Al-Rubeai, M. (1994). Peptone, a low cost growth-promoting nutrient for intensive animal cell culture. Cytotechnology, 16, 17-26.

Jäger, V., Lehmann, J. & Friedl, P. (1988). Serum-free growth medium for the cultivation of a wide spectrum mammalian cells in stirred bioreactors. Cytotechnology, 1, 319-329.

Kao, J., Huey, G., Kao, R. & Stern, R. (1990). Ascorbic Acid Stimulates Production of Glycosaminoglycans in Cultured Fibroblasts. Exp. Mol. Pathol., 53, 1-10.

Kim, E. J., Kim, N. S. & Lee, G. M. (1998). Development of a serum-free medium for the production of humanized antibody from Chinese hamster ovary cells using a statistical design. In Vitro Cell. Dev. Biol., 34, 757-761.

Kim, S. H. & Lee, G. M. (2009). Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Appl. Microbiol. Biotechnol., 83, 639-648.

Keenan, J., Pearson, D. & Clynes, M. (2006). The role of recombinant proteins in the development of serum-free media. Cytotechnology, 50, 49-56.

Keen, M. J. & Rapson, N. T. (1995). Development of a serum-free medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line. Cytotechnology, 17, 153-163.

Kenyon, S. H., Nicolaou, A., Ast, T. & Gibbons, W. A. (1996). Stimulation in vitro of vitamin B12-dependent methionine synthase by polyamines. Biochem. J., 316, 661-665.

Kovář, J. & Franĕk, F. (1989). Growth-Stimulating Effect of Transferrin on a Hybridoma Cell Line: Relation to Transferrin Iron-Transporting Function. Exp. Cell. Res., 182, 358-369.

Lane, D. J. R. & Richardson, D. R. (2014). The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption. Free Radic. Biol. Med., 75, 69-83.

Lee, G. M., Kim, E. J., Kim, N. S., Yoon, S. K., Ahn, Y. H. & Song, J. Y. (1999). Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. J. Biotechnol., 69, 85-93.

Liu, C. H. & Chang, T. Y. (2006). Rational development of serum-free medium for Chinese hamster ovary cells. Process Biochem., 41, 2314-2319.

Lobo-Alfonso, J., Price, P. & Jayme, D. (2010). Benefits and Limitation of Protein Hydrolysates as Components of Serum-Free Media for Animal Cell Culture Applications. Springer Science + Business Media, Berlin.

Mendonça, R. Z. & Pereira, C. A. (1998). Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor. Bioprocess Eng., 18, 213-218.

Merten, O. W., Kallel, H., Manuguerra, J. C., Tardy-Panit, M., Crainic, R., Delpeyroux, F., Van der Werf, S. & Perrin, P. (1999). The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnology, 30, 191-201.

Michiels, J. F., Barbau, J., De Boel, S., Dessy, S., Agathos, S. N. & Schneider, Y. J. (2011). Characterisation of beneficial and detrimental effects of a soy peptone, as an additive for CHO cell cultivation. Process Biochem., 46, 671-681.

Morris, A. E. & Schmid, J. (2000). Effects of Insulin and LongR3 on Serum-Free Chinese Hamster Ovary Cell Cultures Expressing Two Recombinant Proteins. Biotechnol. Prog., 16, 693-697.

Murakami, H., Masui, H., Sato, G. H., Sueoka, N., Chow, T. P. & Kano-Sueoka, T. (1982). Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc. Natl. Acad. Sci. USA, 79, 1158-1162.

Okamoto, T., Tani, R., Yabumoto, M., Sakamoto, A., Takada, K., Sato, G. H. & Sato, J. D. (1996). Effects of insulin and transferrin on the generation of lymphokine-activated killer cells in serum-free medium. J. Immunol. Methods, 195, 7-14.

Parampalli, A., Eskridge, K., Smith, L., Meagher, M. M., Mowry, M. C. & Subramanian, A. (2007). Development of serum-free media in CHO-DG44 cells using a central composite statistical design. Cytotechnology, 54, 57-68.

Petiot, E., Guedon, E., Blanchard, F., Gény, C., Pinton, H. & Marc, A. (2010). Kinetic characterization of Vero cell metabolism in a serum-free batch culture process. Biotechnol. Bioeng., 107, 143-153.

Petiot, E., Fournier, F., Gény, C., Pinton, H. & Marc, A. (2010). Rapid Screening of Serum-Free Media for the Growth of Adherent Vero Cells by Using a Small-Scale and Non-invasive Tool. Appl. Biochem. Biotechnol., 160, 1600-1625.

Quesney, S., Marc, A., Gerdil, C., Gimenez, C., Marvel, J., Richard, Y. & Meignier, B. (2003). Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium. Cytotechnology, 42, 1-11.

Rourou, S., Van der Ark, A., Van der Velden, T. & Kallel, H. (2009). Development of an Animal-Component Free Medium for Vero Cells Culture. Biotechnol. Prog., 25, 1752-1761.

Souza, M. C. O., Freire, M. S., Schulze, E. A., Gaspar, L. P. & Castilho, L. R. (2009). Production of yellow fever virus in microcarrier-based Vero cell cultures. Vaccine, 27, 6420-6423.

Sung, Y. H., Lim, S. W., Chung, J. Y. & Lee, G. M. (2004). Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl. Microbiol. Biotechnol., 63, 527-536.

Taub, M., Chuman, L., Saier, M. H. & Sato, G. (1979). Growth of Madin-Darby Canine Kidney Epithelial Cell (MDCK) Line in Hormone-Supplemented, Serum-Free Medium. Proc. Natl. Acad. Sci. USA, 76, 3338-3342.

Thomas, T. & Thomas, T. J. (2001). Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci., 58, 244-258.

Thommassen, Y. E., Rubingh, O., Wijffels, R. H., Van der Pol, L. A. & Bakker, W. A. M. (2014). Improved poliovirus D-antigen yields by application of different Vero cell cultivation methods. Vaccine, 32, 2782-2788.

Trabelsi, K., Samia, R., Loukil, H., Majoul, S. & Kallel, H. (2006). Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor. J. Biotechnol., 121, 261-271.

Traber, M. G. & Stevens, J. F. (2011). Vitamins C and E: Beneficial effects from a mechanistic perspective. Free Radic. Biol. Med., 51, 1000-1013.

Van der Valk, J., Mellor. D., Brands, R., Fischer, R., Gruber, F., Gstraunthaler, G., Hellebrekers, L., Hyllner, J., Jonker, F. H., Prieto, P., Thalen, M. & Baumans, V. (2004). The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol. in Vitro, 18, 1-12.

Van der Valk, J., Brunner, D., Smet, K. De., Svenningsen, Å. F., Honegger, P., Knudsen, L. E., Lindl, T., Noraberg, J., Price, A., Scarino, M. L. & Gstraunthaler, G. (2010). Optimization of chemically defined cell culture media-Replacing fetal bovine serum in mammalian in vitro methods. Toxicol. in Vitro, 24, 1053-1063.

World Health Organization. (1998). Requirements for the use of animal cells as in vitro substrates for the production of biological. WHO Technical Report Series, 878, 20-55 (Annex 1).

Zhaolie, C., Chengzu, X., Hong, L., Benchuan, W. & Xihua, J. (1996). A novel serum-free medium for the cultivation of Vero cells on microcarriers. Biotechnol. Tech., 10, 449-452.