Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity

Abstract

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 characterized by 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 (micro(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 micro(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 micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during 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 no product 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.

Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production

“…Therefore, if the High-Five cell cycle is synchronized in G 0 and G 1 as a result of the cells' immobilization, they may be more easily infected by the baculovirus and probably more productive (49). Unfortunately, the only existing (and few) data come from Sf-9 infections (46,49), making imperative the need for thorough studies of High-Five cell cycle and its relation to protein production. The exhaustion of glucose by 72 h p.i.…”

Characterisation of Tetraploid and Diploid Clones of Spodoptera frugiperda Cell Line

“…Yet, DNA analysis of the Sf-9 cell line from different international laboratories has revealed that the ploidy levels of this insect cell line vary depending on its culture history and origin (Jarman-Smith et al 2002). Doverskog et al (2000) reported the appearance of an octaploid subpopulation during the G2/M arrest, which was explained as due to the latent possibility of transition of G2/M cells into an endoreplicative state. Other observations (Vaughn et al 1977;Disney and McCarthy 1985) suggested that the chromosomes seem to spontaneously fragment during growth and expansion which raises the question.…”

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

“…The cell density effect has been reported mainly for Sf9 cells Caron et al, 1990;Doverskog et al, 2000;Jesionowski and Ataai, 1997;Radford et al, 1997;Reuveny et al, 1993;Taticek and Shuler, 1997;Wong et al, 1996;Yamaji et al, 1999). There are a few investigations conducted in other cell lines such as Anticarsia gemmatalis (saUFL-AG-286) (Micheloud et al, 2009), Trichopulsia ni (Hi5) (Chico and Jager, 2000;Ikonomou et al, 2004;Taticek and Shuler, 1997;Yang et al, 1996), and…”

“…It has been suggested that the cause of the drop in productivity at high cell densities for baculovirus infection processes is related to nutrient limitations rather than accumulation of toxins (Drews et al, 1995;Radford et al, 1997;Wong et al, 1996). This is supported by the fact that there is an increase in the cell specific yield with an improvement of the nutrient supply through fed-batch strategies Jardin et al, 2007) or fresh medium replacement (Chakraborty et al, 1996;Doverskog et al, 2000;Ikonomou et al, 2004;Jesionowski and Ataai, 1997;Lindsay and Betenbaugh, 1992;Radford et al, 1997;Reuveny et al, 1993). The nutrient limitation may involve micronutrients such as vitamins or trace elements, some of which are cofactors for enzymes involved in critical processes such as catabolism (e.g.…”

“…Recently, the reduction in recombinant protein production at high cell densities has been shown to be strongly correlated with limitations in the upstream processes of virus replication and transcription (Huynh et al, 2013). Other possible factors causing low productivity in high density cultures have been suggested including oxygen limitations (Lecina et al, 2006;Palomares et al, 2004;Taticek and Shuler, 1997), the cell cycle phase at the time of infection (Calles et al, 2006;Doverskog et al, 2000) and the history of the culture .…”