Table 2.

Lay out of the optimization experiments using a central composite design

Experiment no	X1	X2	X3	Cell yield (× 105 cells/well)
1	4.097 (− 1)	0.0692 (− 1)	0.02864 (− 1)	8.20 ± 0.10
2	16.097 (+ 1)	0.0692 (− 1)	0.02864 (− 1)	11.20 ± 0.28
3	4.097 (− 1)	0.2692 (+ 1)	0.02864 (− 1)	10.38 ± 0.13
4	16.097 (+ 1)	0.2692 (+ 1)	0.02864 (− 1)	11.80 ± 0.30
5	4.097 (− 1)	0.0692(− 1)	0.06864 (+ 1)	11.80 ± 0.40
6	16.097 (+ 1)	0.0692(− 1)	0.06864 (+ 1)	11.00 ± 0.25
7	4.097 (− 1)	0.2692 (+ 1)	0.06864 (+ 1)	11.60 ± 0.30
8	16.097 (+ 1)	0.2692 (+ 1)	0.06864 (+ 1)	10.03 ± 0.03
9	0.005 (− 1.68)	0.1692 (0)	0.04864 (0)	10.00 ± 0.10
10	20.189 (+ 1.68)	0.1692 (0)	0.04864 (0)	12.00 ± 0.25
11	10.097 (0)	0.0010 (− 1.68)	0.04864 (0)	10.50 ± 1.00
12	10.097 (0)	0.3374 (+ 1.68)	0.04864 (0)	11.00 ± 1.15
13	10.097 (0)	0.1692 (0)	0.01500 (− 1.68)	11.20 ± 1.20
14	10.097 (0)	0.1692 (0)	0.08228 (+ 1.68)	11.60 ± 0.85
15	10.097 (0)	0.1692 (0)	0.04864 (0)	12.10 ± 0.40
16	10.097 (0)	0.1692 (0)	0.04864 (0)	12.07 ± 0.28
17	10.097 (0)	0.1692 (0)	0.04864 (0)	12.05 ± 0.70

In this study, Vero cells were incubated in SCM in 6-well plates at 37 °C, 5% CO₂ for 3 h and then replaced with tested media. Cells were cultured at 37 °C, 5% CO₂ for 4 days. Cell cultures were carried out in triplicate and expressed as mean ± SD. X1, X2, and X3 were respectively vitamin C; vitamin B12 and recombinant human epidermal growth factor (rEGF) and were expressed as mg/L. Code unit that was designed according to a central composite design was shown in parentheses at-1.68, − 1, 0, + 1, and + 1.68 after its actual concentration