Development of Serum-Free Medium for Spodoptera frugiperda (Sf9) Insect Cell Cultivation and Recombinant Protein Production

Abstract

This research aimed to: 1) develop a serum-free medium (SFM) for Spodoptera frugiperda (Sf9) insect cell culture, 2) study the effect of microfiltration and ultrafiltration of soytone and yeast extract on the growth of Sf9 cells, and 3) examine recombinant protein production in Sf9 insect cells using a developed serum-free medium (OSF9- SFM). Sf9 cells were cultured in Grace's insect cell culture media supplemented with different concentrations of chemically defined lipid concentrate (CDLC), soytone (ST), and yeast extract (YE), using the Central Composite Design method. Low-medium-high levels of substances were assigned as follows: CDLC at 0.5, 0.75 and 1 % (v/v); ST at 3, 7 and 11 g/L; and YE at 3, 7 and 11 g/L. Microfiltration and ultrafiltration of ST and YE fractions were done with 10 and 3 kDa molecular weight cut-off membranes. Fraction #1 (crude ST or YE), fraction #2 (≤ 10 kDa ST or YE), and fraction #3 (≤ 3 kDa ST or YE) were formulated for OSF9-SFM. Sf9 cells were transfected with plasmid pEGFP-N1 encoding recombinant green fluorescent protein (GFP) and infected with recombinant-baculoviruses (r-baculoviruses) generated by recombinant-Bacmid (rBacmid) containing anti-hepatitis B virus surface antigen (anti-HBsAg). The effect of OSF9-SFM on recombinant protein production in Sf9 cells was determined using immunofluorescence assay (IFA). The optimum concentrations of CDLC, YE, and ST in OSF9-SFM were 0.5% (v/v), 11 g/L and 3 g/L, respectively, in which 10-kDa ultrafiltration fractions of YE and ST were used. The maximum cell density obtained from adherent culture was 7.17 x 10[superscript5] cells/mL, whereas cell numbers from suspension culture reached 2.17 x 10[superscript6] cells/mL with a specific growth rate of 0.027 h[superscript-1]. The average GFP cells in the medium supplemented with serum peaked at 34 cells/well, while SF900 II and OSF9-SFM fluorescence was only 13 and 2 cells/well, respectively. Recombinant-Bacmid was unable to transfect Sf9 cells in OSF9-SFM, possibly due to the hydrolysate present in the media inhibiting transfection of r-Bacmid. However, Sf9 cells grown in OSF9-SFM could be effectively infected with passage 2 of r-baculoviruses derived from SF900 II culture, and an expression of anti-HBsAg fluorescence signals was clearly detected by IFA. The development of in-house OSF9-SFM will continue to increase both cell numbers and protein expression in the next step.