Abstract
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™ soytone and Bacto™ 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™ yeast extract (7.0 g/L) combined with various concentrations of Bacto™ 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™ soytone (7.0 g/L) combined with various concentrations of Bacto™ yeast extract (0.1-7.0 g/L) in SFM01-M were used. However, the combination of Bacto™ soytone at 0.1 g/L and Bacto™ yeast extract at 7.0 g/L or Bacto™ soytone at 7.0 g/L and Bacto™ 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™ soytone, Bacto™ 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™ soytone (0.1 g/L), Bacto™ 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.