Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implicatons for proliferation and productivity

摘要

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterizedby an accumulation of 50-70% of the cells in the G_2/M phase of the cell cycle during the first 24 h after inoculation. following the cell cycle arrest, the cell population was redistributed into G_1 and in particular into the S phase. Maximum rate of proliferation (#mu#N, max) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G_2/M phase. The following declining #mu#N could be explained by a slow increase in the G_2/M cell population.However, at approximately 100 h, an abrupt increase in the amount of G_2/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G_2/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase #mu#N, max, decrease the time to reach #mu#N, max, and decrease the synchronization of cells in G_2/Mduring the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of #beta#-galactosidase up to 68-75 h of culture. Beyond this point almost noproduct was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G_2/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G_2/M cell cycle arrest.