No abstract

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.

meu orientador e amigo, que me recebeu tão bem e me ensinou a pensar como uma pesquisadora, transformando o trabalho em momentos de aprendizagem regados com paciência e dedicação. Ao Dr. Carlos Augusto Pereira, pela contribuição de infra-estrutura e também pelos momentos de reflexão. A Profa. Dra. Soraia Attie Calil Jorge por tantos ensinamentos em biologia, passados com muito carinho e amizade. A Profa. Dra. Maria Cândida R. Facciotti por todos os ensinamentos e atenção a qualquer hora. A Profa. Dra. Elizabeth Augusto pelos momentos de discussão e troca de idéias que tanto me ensinaram. Ao Dr. Ronaldo Zucatelli Mendonça pelo apoio e amizade em todos os momentos. Aos maravilhosos amigos: Isabel, Marcos, Alexandra e Mariza por todos os ensinamentos, apoio e amizade que tornaram o dia-a-dia mais prazeroso.

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.

minha co-orientadora e amiga, pelos bons momentos de discussão.Ao Dr. Carlos Augusto Pereira, pela contribuição de infraestrutura e tantos ensinamentos, conselhos e apoio.A Profa. Dra. Soraia Attie Calil Jorge por tantos ensinamentos em biologia, passados com muito carinho e por sua amizade verdadeira.Ao Dr. Renato Astray por boas discussões biológicas e por sempre me apoiar e incentivar.Ao meu amigo-filho Marcelo Aguiar por todo carinho, amizade e pelos ensaios! Aos meus amigos-filhos Daniella, Marcos e Cássia por toda amizade, troca de ideias e por me apoiarem e me ajudarem.