Study of breathing activity of wild and transfected line of insect cells through cultivations in bioreactors. / Estudo da atividade respiratória de linhagens selvagens e transfectadas de células de insetos através de cultivos em biorreatores.

AUTOR(ES)

Marilena Martins Pamboukian

DATA DE PUBLICAÇÃO

2007

RESUMO

Specific respiration rate (QO2) 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 QO2 and critical oxygen concentration (Ccrit). Wild Spodoptera frugiperda (Sf9) and wild and transfected Drosophila melanogaster S2 cells (able to produce different proteins) were used. All experiments were performed in 1-liter working volume Inceltech bioreactor, maintaining temperature controlled at 28ºC, agitation rate at 100 rpm, and dissolved oxygen (DO) at 40% of air saturation, through membrane diffusion of mixed gases (O2 and N2) at constant total flow rate. SF900II serum free medium was used. QO2 was measured through dynamic method and oxygen mass balance in the liquid phase. In this work a new process was implemented during the dynamic method to interrupt completely the oxygen transfer during the execution of this method. It was also implemented a methodology for measurement of Ccrít (determined when DO reduces its decay rate, without oxygen transfer). Maximum cell concentration (Xm), maximum specific respiration rate (QO2) in the exponential phase and Ccrít were reached, as follows: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfected for GPV expression): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfected for EGFP expression): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfected for HbsAg expression): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. From these results, it can be concluded that the studied cell lines have different respiration activity and the new developed methodologies behave satisfactorily.

ASSUNTO(S)

drosophila melanogaster biorreatores spodoptera frugiperda drosophila melanogaster insect cells respiração celular spodoptera frugiperda bioreactors cell respiration células de insetos

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