Physiology and productivity of serum-free Spodoptera frugiperda Sf9 insect cell cultures

Detta är en avhandling från Stockholm : KTH

Sammanfattning: The objective of this study was to investigate the mechanisms and factors controlling growth and proliferation in serum-free Spodoptera frugiperda Sf9 cultures as well as the implications of these factors for protein production in the baculovirus expression system.The physiology of recently thawed, low passage (Lp) Sf9 cultures, were compared to high passage (Hp) cultures at p>100. Lp cells passed a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag-phase and an increased maximum specific proliferation rate, µN,max, from 0.03/h to 0.04/h. Conditioned medium (CM), 10 kDa CM filtrate and 10 kDa CM concentrate promoted proliferation of Lp Sf9 cells, but had no effect after the switch. Sf9 cell cycle dynamics were characterized by an initial G2/M arrest, which synchronized the cells and this feature was more pronounced for Hp than for Lp cells. CM addition decreased the initial arrest for Lp cultures, but did not affect Hp cells. Late in the culture, a final G2/M accumulation occurred. An octaploid population emerged during G2/M arrests. Further, a 49 kDa proform and a 39 kDa active form of Sf9 cathepsin L was identified from gelatine zymography of Sf9 CM, on basis of inhibitor profile and substrate range. Removal of procathepsin L during the course of an Sf9 culture had a negative effect on Sf9 proliferation. Procathepsin L was also identified in Trichoplusia ni High five CM. High five CM promoted growth of Sf9 cells, but when procathepsin L was removed no effect was observed. It is suggested that these observations are due to an autocrine system controlling proliferation. One Sf9 mitogen might be a <10 kDa peptide, while the effect of 10 kDa CM concentrate may originate from procathepsin L. A hypothesis is therefore that procathepsin L acts as a mitogen in Sf9 cultures, perhaps in concert with the <10 kDa peptide.The volumetric product yield (P) in baculovirus infected Sf9 cells increased linearly up to 68-75 h of culture. Beyond this point almost no product was detected. Medium renewal at infection prolonged the productivity phase until 117 h, but generated only a 10% increase in P. The specific product formation rate (YP/N) was highest at µN,max. YP/N of Lp cells decreased by 30-50% when 20% CM or 10 kDa CM filtrate was added, whereas addition of CM to cells having passed the switch on growth kinetics did not affect productivity. Further, Hp cells exhibited a two-fold higher YP/N than Lp cells, when infected during the initial 48 h of culture. This coincided with a high degree of synchronization. Yeastolate limitation was used to achieve artificial synchronization of an Lp culture, and YP/N could thereby be maintained high during a prolonged time, resulting in a 69% increased P. This suggests that a decreasing degree of synchronization during the course of a culture partly explains the cell-density dependent drop in productivity in Sf9 cells.Finally, ~10 kDa gel filtration fractions from Sf9 and High five CM were found to be bactericidal. Exposure of a Bacillus megaterium culture for eight min to an Sf9 CM fraction killed 99% of the population, and 60 min exposure killed 35% of an Escherichia coli population. In both cases cell lysis was observed. B. megaterium incubated in an High five CM fraction lost 97% viability in 40 min. The effect of the High five CM fraction most probably originated from a lysozyme precursor protein, whereas the Sf9 executor remains unknown.