Optimized Insect Cell Culture for the Production of Recombinant Heterologous Proteins and Baculovirus Particles

Pereira, Carlos Augusto; Pouliquen, Yann; Rodas, V.; Massotte, Dominique; Mortensen, C.J.; Sogayar, Mari Cleide; Murcia, Josiane Ménissier‐de

doi:10.2144/01316bm07

Cited by 15 publications

(11 citation statements)

References 23 publications

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“…The virion containing -PIBs are ingested by insect larvae as food contaminants and after digestion in the insect midgut, virions may replicate in midgut cells and attain other tissues, leading to larvae disintegration, which becomes a fluid sac containing large amounts of PIBs. The AgMNPV PIBs, commercially produced in farmer's field, are currently used as bioinsecticide against the velvetbean caterpillar Anticarsia gemmatalis, and studies of cell cultures derived PIBs production in industrial scale are envisaged (Moscardi 1999;Pereira et al 2001).…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, only 1.4 out of 21 million soybean hectares are today treated in Brazil with the commercially available AgMNPV bioinsecticide. Among the drawbacks to foster the Ag-MNPV production and application, one includes the technical and economical difficulties for PIBs production with a cell culture based technology (Bonning et al 1995;Pereira et al 2001;Lua et al 2002). Currently the production is conducted in farmer's field and lead to a cost for the farmer lower than the cost of chemical insecticides.…”

Section: Introductionmentioning

confidence: 99%

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Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

et al. 2005

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We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k L a of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5 · 10 6 cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the lX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day À1 ). The glucose consumption and lactate production were higher in the infected cultures (lGlucose and lLactate of, respectively, 0.0248 and 0.0089 · 10 À8 g/cell · day in infected cultures and 0.0151 and 0.0046 · 10 À8 g/cell · day in non infected ones). The glutamine consumption did not differ in both cultures (lGlutamine of 0.0034 and 0.0037 · 10 À8 g/cell · day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10 6 to 2 · 10 6 cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL 50 . Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL 50 . The loss Cytotechnology (2005)

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

et al. 2005

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“…A stock of recombinant SIV Gag p55 protein was prepared utilizing the baculovirus system essentially as described elsewhere (45). The resulting protein was analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain a single band of 55 kDa, which showed reactivity against a monoclonal antibody against SIV Gag.…”

Section: Animalsmentioning

confidence: 99%

Evidence for Antibody-Mediated Enhancement of Simian Immunodeficiency Virus (SIV) Gag Antigen Processing and Cross Presentation in SIV-Infected Rhesus Macaques

Villinger¹,

Mayne²,

Boštík³

et al. 2003

J Virol

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By using the dominant simian immunodeficiency virus (SIV) Gag Mamu-A01 restricted major histocompatibility complex (MHC) class I epitope p11CM, we demonstrate antibody-mediated enhanced MHC class I cross presentation of SIV Gag. In vitro restimulation of peripheral blood mononuclear cells from SIV-infected rhesus macaques with recombinant full-length SIV Gag p55 plus p55 affinity-purified immunoglobulin G (p55 Gag/p55-IgG) led to the generation of markedly higher frequencies of p11CM specific precursor cytotoxic T lymphocytes (p-CTLs) compared with restimulation with (i) SIV Gag p55 alone or (ii) optimal concentrations of the p11CM peptide alone. These results, along with the finding that CD4 depletion abrogated the enhancement, suggest a prominent role for CD4 ؉ T cells. Testing for p-CTLs against other Mamu-A01-restricted SIV Gag epitopes suggested that this mechanism favored recognition of the dominant p11CM peptide, potentially further skewing of the CTL response. The p-CTL enhancing effect was also decreased or abrogated by pepsin digestion of the p55-specific IgG or by the addition of monoclonal antibodies to Fc receptor (FcR) II/III, suggesting that the effect was dependent on FcR-mediated uptake of the immune-complexed antigen. Finally, incubation of antigen-presenting cells with SIV Gag p55 immune complexes in the presence of lactacystin or of bafilomycin indicated that the mechanism of antibody-mediated enhancement of cross presentation required both the proteasomal and the endosomal pathways. These data demonstrate for the first time the cross presentation of antigens via immune complexes in lentiviral infection and indicate a heretofore-unrecognized role for antibodies in modulating the magnitude and potentially also the breadth of MHC class I-restricted antigen processing and presentation and CTL responses.

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“…They show, for instance, a good resistance to shear stress as well as to pH variations and as a consequence the culture procedures can be based on technologies currently used for animal cells, such as tissue culture flasks and spinners bottles and, in addition, can also be performed in orbital shaker bottles and on simpler media (Weiss and Vaughn 1986;Mitsuhashi 1998;Pereira et al 2001;Weber et al 2002).…”

Section: Introductionmentioning

confidence: 99%

Insect cells respiratory activity in bioreactor

et al. 2008

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Specific respiration rate (Q O 2 ) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure Q O 2 . Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 9 10 6 cells/mL and maximum specific respiration rate (Q O 2 max ) of 7.3 9 10 -17 molO 2 /cell s. Drosophila melanogaster (S2) cells achieved 51.2 9 10 6 cells/mL and Q O 2 max of 3.1 9 10 -18 molO 2 /cell s. S2AcGPV (expressing with rabies virus glycoprotein) reached 24.9 9 10 6 cells/mL and Q O 2 max of 1.7 9 10 -17 molO 2 /cell s, while S2MtEGFP (expressing green fluorescent protein) achieved 15.5 9 10 6 cells/mL and Q O 2 max = 1.9 9 10 -17 molO 2 /cell s. Relating to the Sf9, S2 cells reached higher maximum cell concentrations and lower specific respiration rate, which can be explained by its smaller size. These results presented useful information for scale-up and process control of insect cells.

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Optimized Insect Cell Culture for the Production of Recombinant Heterologous Proteins and Baculovirus Particles

Cited by 15 publications

References 23 publications

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

Evidence for Antibody-Mediated Enhancement of Simian Immunodeficiency Virus (SIV) Gag Antigen Processing and Cross Presentation in SIV-Infected Rhesus Macaques

Insect cells respiratory activity in bioreactor

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