High Cell Density in Insect Cell Cultures (original) (raw)

Elsevier eBooks, 1994

Abstract

Summary Spodopdera frugiperda (Sf-9) cells were propagated in batch, fed-batch, perfusion and medium replacement methods, achieving cell concentrations of 4.5, 6.7, 9.5 and 11.6×106 cells/ml respectively. Fed-batch cultures were carried out by continuous feeding of the culture with glucose, glutamine and yeastolate ultrafiltrate. Perfusion cultures were carried out by continuous medium perfusion at a rate of 0.4–0.5 vol/vol day using an external 0.2μm pore size hollow fiber microfilter for continuous cell-medium separation. In the medium replacement method the same hollow fiber microfilter was used for replacement of 80% of the spent medium with fresh medium when growth limiting conditions occured. Although the highest cell density was achieved by applying perfusion or periodical replacement of the medium, better medium utilization (6.7×106 cells per ml medium) was obtained in the fed batch methods. We have combined the medium replacement and fed-batch methods, in order to achieve both, high medium utilization (6.7×106 cells per ml medium) and high cell concentration (16×106 cells/ml). Maximal production of recombinant proteins (0.9 mg of E. coli b-galactosidase or 1.7 ug of glucocerebrosidase per 106 cells) in batch cultures was achieved when the Sf-9 cells were infected with the recombinant baculoviruses in the early or middle exponential phase of growth. Less than 10% of this productivity was obtained when cells were infected at the late exponential phase (cell concentration of about 4×106 cells/ml). Therefore, in order to obtain the maximal protein productivity, cells from the late exponential phase of growth were resuspended with fresh growth medium prior to the viral infection and fed with glucose glutamine and yeastolate ultrafiltrate during the infection. These conditions do not yield the maximal protein productivity when viral infection is done at cells concentration above 4×106. A continuous two stage reactor system for production of recombinant proteins is suggested. In the first stage the Sf-9 cells are propagated to a concentration of 16×106 cells/ml using the medium replacement-fed batch method. Every 4 days, 95% of the cell culture is transferred to the second stage reactor where it is diluted with fresh growth medium to cell concentration of 4×106 cells/ml. During the infection, the culture is continuously fed with nutrients.

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