Development of a modified serum-free medium for Vero cell cultures: effects of protein hydrolysates, l-glutamine and SITE liquid media supplement on cell growth

Vero cells have been widely used in the viral vaccine production due to the recommendation of the World Health Organization regarding its safety and non-tumorigenicity. The aim of this study was to describe the development a modified serum-free medium for Vero cell cultures. Two protein hydrolysates (Bacto (TM) soytone and Bacto (TM) yeast extract), vitamin C, vitamin B12, SITE liquid media supplement, and recombinant human epidermal growth factor (rEGF) were investigated as serum substitutes. A sequential experiment of fractional factorial and central composite design was applied. A modified serum-free medium obtained (named as SFM01-M) was verified. Contrary to P0, the cell yields obtained at P1, P2, and P3 decreased continuously during the verification experiments indicating that Vero cells could not adapt to SFM01-M as expected according to the empirical mathematical model. To improve cell growth after P0, protein hydrolysates, l-glutamine, and SITE liquid media supplement were further investigated. The results showed that cell yields gradually decreased from P1 to P3 when a fixed concentration of Bacto (TM) yeast extract (7.0 g/L) combined with various concentrations of Bacto (TM) soytone (0.1-7.0 g/L) in SFM01-M were used. Similarly, cell yields also gradually decreased from P1 to P3 when a fixed concentration of Bacto (TM) soytone (7.0 g/L) combined with various concentrations of Bacto (TM) yeast extract (0.1-7.0 g/L) in SFM01-M were used. However, the combination of Bacto (TM) soytone at 0.1 g/L and Bacto (TM) yeast extract at 7.0 g/L or Bacto (TM) soytone at 7.0 g/L and Bacto (TM) yeast extract at 0.1 g/L in SFM01-M could give the maximum cell yield at P3 when compared with other combinations. In addition, the addition of SITE liquid media supplement (0.1-2.0% v/v) in SFM01-M in which the concentrations of Bacto (TM) soytone, Bacto (TM) yeast extract, and l-glutamine were fixed at 0.1 g/L, 0.1 g/L, and 4.0 mM, respectively, the results showed that the cell yields obtained at P3 were not significantly different. From this study, the optimum concentrations of SFM01-M components were as follows: Bacto (TM) soytone (0.1 g/L), Bacto (TM) yeast extract (0.1 g/L), vitamin C (9.719 mg/L), vitamin B12 (0.1725 mg/L), SITE liquid media supplement (0.1-2.0% v/v), rEGF (0.05756 mg/L), l-glutamine (4.0 mM), MEM non-essential amino acids (1.0% v/v), sodium pyruvate (1.0 mM), MEM (9.4 g/L), and sodium hydrogen carbonate (2.2 g/L). However, to evaluate SFM01-M in the long-term subculture of Vero cells, the efficiency of SFM01-M will be further investigated.

Automated summary

The study described the development of a modified serum-free medium (SFM01-M) for Vero cell cultures, exploring various serum substitutes and conducting verification experiments. The optimal cell yield was achieved under specific combination conditions.