Evaluation of concentrated milk whey as a supplement for SF9 Spodoptera frugiperda cells in culture
Fabiana R. X. Batista
Carlos A. Pereira
Ronaldo Z. Mendonça
Angela M. Moraes*
Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil), Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP grant no. 02/09482-3, São Paulo, Brazil) and Butantan Foundation (São Paulo, Brazil). C.A. Pereira is recipient of a fellowship from the Conselho Nacioanl de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil).
Keywords: baculovirus, concentrated milk whey, Grace's medium, insect cells, media formulation, Spodoptera frugiperda Sf9.
In the last decade the insect cell/baculovirus system has been widely used to produce wild-type baculoviruses as biopesticides and for the abundant expression of recombinant proteins. Recently, in several countries, such as Brazil, baculovirus-based pesticides obtained through the infection of velvet bean caterpillar larvae were not used in larger quantities simply because of their limited supply. Since this tendency is increasing worldwide, causing significant economical and environmental impact, strategies that allow the production of baculovirus at lower costs and higher amounts are particularly interesting. Approaches based on the infection of in vitro cultured insect cells with baculovirus may be economically attractive if culture media costs can be reduced, provided that virus infectivity is maintained after successive cultures.
Insect cell culture can be performed in basal media, such as Grace's medium supplemented with 5% or 10% (v/v) serum, most usually fetal bovine serum (FBS). However, problems such as high cost, lot-to-lot inconsistency, introduction of adventitious agents and presence of proteins may limit FBS widespread use in the insect cell/baculovirus system. Therefore, many studies have been performed during the last years aiming to reduce or eliminate serum requirements from insect cells, resulting in several effective serum-free formulations available in the market. On the other hand, in spite of serum-free media such as Sf900II being capable to support high cell densities, frequently their costs are too high when large scale baculovirus polyhedra production is desired.
In Brazil, due to the extensive alcohol production by fermentation and cheese processing, residues such as yeast extract and milk whey are readily available, and could be used as inexpensive and highly nutritive insect cell culture media supplements. Yeast extract contains amino acids, peptides, polysaccharides, vitamins, nucleic acids, lipids and traces of important metals that can promote Sf9 cell growth. Among other components, milk whey contains proteins, lactose and lipids, being much less complex than FBS and, therefore, easier to be standardized when required, what motivated the present study.
The aim of this work was, therefore, the evaluation of supplements to Grace's medium, partially substituting FBS, intending to increase the productivity of Spodoptera frugiperda Sf9 cells and also of baculovirus. The effects of glucose, Pluronic F68 (PF68), yeast extract (YE), milk whey concentrated by ultrafiltration (MWR) and FBS on viable cell concentration in stationary and midexponential phase, on cell viability in the midexponential phase, and also on specific cell growth rate, were evaluated.
All experiments were carried out in 100 mL shake flasks (with working volumes of 15 ml) inoculated with 2.5 x 105 viable cells.ml-1 and incubated in a rotary shaker at 100 rpm and 28ºC. The statistical factorial design strategy was employed throughout the work and Grace's medium containing 10% (v/v) FBS was used as a control, as well as the Sf900II serum-free medium.
The achieved results show that glucose concentration and PF68 percentage did not significantly affect cell growth and viability in the evaluated ranges (1.8 to 2.7 g.l-1 for glucose and 0.0 to 0.2 for PF68). Glucose was depleted and lactate was produced, however, after two days lactate was consumed simultaneously with glucose, being considered as an alternative carbon source.
The addition of concentrated whey protein and yeast extract, on the other hand, significantly increased cell concentration and also provided viral production comparable to that achieved in Sf900II medium at a much lower cost. After full adaptation of the cells to grow in shake flasks using selected media formulations, viable cell concentrations in stationary phase up to 37.5 x 106 cells.ml-1 were attained. Baculovirus infection of Sf9 cells grown a particular medium formulation (named 4A), consisting of Grace's medium containing 5% MWR, 8 g.l-1 YE, 1% FBS and 2.7 g.l-1 glucose, resulted in up to 1.6 x 107 polyhedra.ml-1. This concentration is 2.6 higher than that obtained with Grace's medium supplemented with 10% of FBS and only 0.9 lower than that observed with the Sf900II serum-free medium. Formulation 4A proved to be interesting not only concerning virus production, but also economically. Besides, differently from Sf900II, it presents a known formulation.
Therefore, the evaluated milk whey derivative is an effective supplement to Grace's medium, together with yeast extract and low amounts of FBS and glucose, to obtain viral products at lower costs. Qualitative constraints concerning viral infection have to be addressed, however, for effective biopesticide production. Provided that the costs of milk whey derivatives are reduced, so will be the final baculovirus cost.