Navegando por Palavras-chave "Animal Cells"

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    Uso do modo de operação em contínuo para avaliar aspectos fisiológicos de linhagem recombinante de drosophila melanogaster
    (Universidade Federal de São Paulo (UNIFESP), 2019-02-19) Oliveira, Carla Reis [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Glycoproteins are complex molecules produced preferentially by animal cells, but their quality pattern rely on the knowledge of cell metabolism and requires engineering tools to ensure greater uniformity and productivity. As long as reports on the metabolism of insect cells are scarce, researches on the physiological aspects of Drosophila*melanogaster are of utmost importance to promote their usage. In order to contribute on the knowledge of this metabolism, the present work aimed to identify growth limiting and inhibitory factors of S2AcRVGP2, a D.*melanogaster S2 cell line stably transfected for the synthesis of Rabies virus glycoprotein (GPV). This study was conducted using TC100 based medium in spinner flasks and in benchtop bioreactor stirred at 100 rpm and with bubbleLfree aeration system. Exploratory cultures on batch mode were carried out with different concentrations of LLcystine (CYS2) and magnesium (Mg2+). The influence on the metabolism of different glucose concentrations (GLC) in the feed medium was studied in a continuous mode. It has been shown that lactate and ammonium did not seem to be growth inhibiting. The supplementation with CYS2 11 mg/L enhanced GPV expression by 75% in batch with initial supplementation and 21% in batch with pulse addition at the end of exponential phase. The evaluation of Mg2+ influence on cell growth kinetic parameters showed that when cultivated with half of its basal concentration, cell production increased 37%, but when cultured on [Mg2+] inferior to 121 mg/L, it limited the growth rate and altered cell membrane morphology. Continuous culture with glucose concentration above 3.75 g/L in the feed medium indicated overflow on glycolysis with consequently limitation of cell growth. The metabolism shifted to a more balanced state when fed with glucose concentration under 2.5 g/L. Cell production increased by 40% and GPV expression doubled in the lowest glucose concentration evaluated (1.25 g/L).