Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

Abstract

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2Ac-GPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L -1 yeastolate ultrafiltrate, 10 g Lmethionine and 1% (v/v) lipid emulsion, reaching 19 9 10 6 cells mL -1 . Maximum specific growth rate and cell productivity were 0.025 h -1 and 0.57 9 10 5 cells mL, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L -1 , however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.

Behavior of Wild-type and Transfected S2 Cells Cultured in Two Different Media

“…The cells maintained in Sf900 II® produced up to 545 mg L −1 of ammonia, while in supplemented supports the general observation that they consist on a robust cell expression system [20]. As shown in Table 2, µ max values were 0.038 h −1 and 0.030 h −1 for S2AcRVGP2k cells cultured in supplemented IPL-41 and Sf900 II® media, respectively.…”

“…These include human plasminogen [14] and mouse endostatin [15]. Non-secreted and secreted recombinant proteins such as the G glycoprotein from rabies virus (RVGP) and the surface antigen of hepatitis B virus (HBsAg), respectively, can be expressed in Drosophila Expression System (DES), both showing to be appropriately processed and biologically active [13,[16][17][18][19][20][21][22][23]. Furthermore, S2AcRVGP2 cells cultivated in IPL-41 medium supplemented with 10 g L −1 glucose, 0.5 g L −1 fructose, 2 g L −1 lactose, 3.5 g L −1 glutamine, 0.6 g L −1 tyrosine, 1.48 g L −1 methionine, 6 g L −1 yeastolate ultrafiltrate, 1% chemically defined lipid concentrate, and 0.1% Pluronic F-68 are able to produce up to 31 ng RVGP L −1 in batch culture provided that dissolved oxygen and pH are controlled at 40% and 6.3, respectively [24].…”

“…Moreover, regulatory agencies such as the European Medicine Evaluation Agency and the Food and Drug Administration (USA) encourage biological compounds manufacturers to reduce or eliminate the use of substances of animal origin [30]. Serum-free medium can be successfully formulated by supplementing basal media with yeastolate and a lipid emulsion (containing cholesterol, alfatocopherol acetate, fatty acids, Tween 80, and Pluronic F-68), among other components [5,20].…”

A high-throughput template for optimizingDrosophilaorgan culture with response-surface methods

“…Of the media tested, M3, Schneider's and D22 are frequently used for the maintenance of Drosophila lines (Echalier, 1997). IPL-41, TC-100 and Sf900 II have been successfully used in the cultivation of Drosophila S2 cells (Batista et al, 2008;Bovo et al, 2008). The media-blending experiment was designed as an efficient method to search the parameter space of concentrations of essential nutrients such as amino acids, salts, sugars and vitamins by varying the relative proportions of the basal media in the culture medium mixture.…”

Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations

“…Several complex glycoproteins were already expressed in the S2 cell system, using these promoters (Mallender et al 2001;Zhang et al 2007;Scotter et al 2006;Brillet et al 2006;Kim et al 2005;Johansson et al 2007;Jennings et al 2006;Li et al 2005;Lim et al 2004;Lee et al 2007). Aiming the expression of high levels of RVGP under the control of these promoters in the S2 cell system, for vaccination as well as structure/function evaluation, many studies were already carried out on cell growth and heterologous recombinant protein expression kinetics (Yokomizo et al 2007;Galesi et al 2008;Swiech et al 2008a;Batista et al 2009;Ventini et al 2010;Lemos et al 2009), as well as on metabolism and synthesis of secondary products (Swiech et al 2008b, c) and culture medium formulation and supplementation (Galesi et al 2007;Batista et al 2008Batista et al , 2011Mendonça et al 2008Mendonça et al , 2009. For constitutive RVGP expression using the actin promoter, instead of a gradual and sustained increase of RVGP, we have observed a sharp RVGP increase, at the beginning of the stationary cell growth phase, which could not be associated with the culture system or the cell culture media, pH, oxygen concentration or substrate change (Galesi et al 2008;Ventini et al 2010;Batista et al 2011).…”