PROTEIN COMPLEXES continued
confirm that the uninfected insect cell lines can be successfully cultured and maintained in an automated micro bioreactor system (Figures 2 and 3).
Figure 2 – Growth of Sf21 cells in ambr bioreactors cultured in triplicate GIBCO and Hyclone Medium.
A subsequent viral infection study of the cells using 12 ambr vessels and three shake flasks seeded with Sf21 cells and cultured for up to eight days was then carried out. Cultures were diluted 2:1 to achieve target cell density for virus infection. The ambr insect cell cultures were infected with a single baculovirus expressing a His-tagged 2-protein complex labeled with YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein). Except for uninfected negative controls (condition A), the ambr system automatically seeded insect cell cultures, cell densities being 1.5 (D), 3.0 (C), or 6.0 (B) × 106 cells/mL, respectively, with the same virus such that the multiplicity of infection (MOI) remained the same in all bioreactors. The shake flask cell cultures were infected with a single baculovirus expressing a His-tagged 2-protein complex labeled with YFP and CFP at 1.5 (D, data unavailable), 3.0 (C), and 6.0 (B) × 106 cells/mL. Increasing YFP and CFP expression detected in both the manual shake flask and automated ambr culture systems over days 1–4 postinfection indicated successful viral infection (Figure 4).
Following analysis of recombinant protein production in this study, the manual shake flask and automated ambr culture systems both indicated that YFP and CFP were produced in all the conditions tested (except the negative control), confirming successful infection (Figure 5). Indeed, the fact that the negative ambr control cultures (condition A) remained uninfected indicates that no viral cross-contamination occurred. Increased protein ex- pression was observed from cultures grown in the ambr bioreactors infected at lower cell densities, and an infection cell density of 1.5 × 106 cells/mL was found to be near optimal. This is again confirmed by the shake flask cultures, with stronger expression observed from cultures infected at lower densities, matching earlier shake flask studies which suggested that infection between 0.5 and 2.0 × 106 cells/mL is optimal for insect cell protein expression.
Figure 3 – Growth of Hi5 cells in ambr bioreactors cultured in triplicate GIBCO and Hyclone Medium.
The observed results confirm that sf21 insect cells can be successfully grown, maintained, and infected in the ambr bioreactors to express multiprotein complexes. The results also suggest that protein expression can be rapidly obtained with reduced effort, enabling speedy assessment of variants and
Figure 4 – Growth of Hi5 cells in ambr bioreactors cultured in triplicate GIBCO and Hyclone Medium. AMERICAN LABORATORY • 34 • SEPTEMBER 2013
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