A. N. Emery

Bcl‐2 over‐expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells

Recent studies have suggested that Bcl-2 can affect cell cycle re-entry by inhibiting the transition from G0/G1 to S phase. In this study, we have taken a novel route to the study of the relationship between Bcl-2 expression and cell cycle progression. Continuous cultures of pEF (control) and Bcl-2 transfected murine hybridoma cells were operated at a range of dilution rates from 0.8 day-1 down to 0.2 day-1. The specific growth rate of the pEF cell line was the same as the dilution rate down to a value of 0.6 day-1. However, as the dilution rate was reduced stepwise to 0.2 day-1, the growth rate levelled-off at approximately 0.55 day-1 and this coincided with a fall in culture viability. By contrast, the specific growth rate of the Bcl-2 transfected cell line followed the dilution rate down to a value of 0.3 day-1 with high levels of cell survival. At high dilution rates, the cell cycle distributions were very similar for both cell lines. However, the distributions diverged as the dilution rate was reduced and, at a rate of 0.2 day-1, the percentage of G1 cells in the Bcl-2 culture was 80%, compared to only 56% in the pEF cell population. This corresponded with a greater extension in the duration of the G1 phase in the Bcl-2 cells, which was 1.7 days at the lowest dilution rate tested, compared to only 0.6 day for the pEF cell line. The durations of the G2/M and S phases remained constant throughout the culture. The maximum doubling time was 1.2 days in the pEF culture compared to 2.3 days in the Bcl-2 culture. Analysis of amino acids, ammonia and lactate concentrations indicated that the observed effects on cell cycle dynamics were probably not due to differences in the culture environment. It is suggested that the expression of Bcl-2 can effect G1 to S phase transition in continuously cycling cells, but this is only apparent at suboptimal growth rates.

Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus

Insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. Initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Spodoptera frugiperda (Sf9) cells. The percentage of cells infected with wild and recombinant baculovirus AcNPV and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were in when infected and by the multiplicity of infection (MOI). However, the cell density at the time of infection and the medium condition showed little direct influence on infectivity. There may, however, be an indirect influence in that these factors determine the relative distribution of cells in the cell cycle. The infectivity is then in turn affected by the relative frequency of cells in the G1, S and G2/M phases. Insect cell specific oxygen uptake rates (1.3-3.4 x 10(-17) mol per cell per s) were essentially similar to or less than those measured for hybridoma cells. However, when Sf9 cells were infected with baculovirus, the specific oxygen uptake rate increased by up to 40%.

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.

Influence of Agitation and sparging on the growth rate and infection of insect cells in bioreactors and a comparison with hybridoma culture

Sf9 insect cells have been cultivated in paired agitated 1.4 L bioreactors with and without Pluronic F‐68 and with and without sparging. The results are reported and, as appropriate, compared to those of hybridoma culture. Without sparging, the cells grew well at agitator speeds from 100 to 400 rpm (equivalent to a realistic range of specific energy dissipation rates). At the higher speeds, a small reduction in growth rate and maximum viable cell concentration was observed. This reduction was shown not to be dependent on the energy dissipation rate but to be due to bubble entrainment. These results are rather similar to those found with hybridoma cells. However, unlike hybridoma cells which grow like unsparged controls when sparged at 0.007 vvm, these insect cells at such a sparge rate show a significant reduction in growth rate and in maximum cell number. This reduction due to the presence of bubbles, whether from head space entrainment or from sparging, could be eliminated by the presence of Pluronic F‐68. These insect cells have then also been infected with recombinant or wild type AcNPV. Agitation had no effect on the rate of infection, but higher levels of MOI enhanced it. Both findings support a diffusion mechanism as previously. The presence of Pluronic F‐68 did not have any impact on the rate of infection and was able to prevent cell damage while agitating and sparging infected cells.

Apoptosis and its control in cell culture systems

Apoptosis is a form of programmed cell death which exhibits highly distinctive morphology. Research activity in this area has increased substantially in recent years, primarily due to the realisation that disregulation of apoptosis is involved in the development of a number of pathological conditions, including cancer and AIDS. However, it is now clear that apoptosis also represents the dominant form of cell death during the culture of industrially important cell lines. This review focuses on the induction of apoptosis during industrial cell cultures as well as the effects of the apoptosis suppresser gene bcl-2 on cell survival in conditions relevant to bioreaction environments. We also present new data which demonstrates that bcl-2 can protect cells from apoptosis induced by oxygen deprivation, a finding which has important implications for large scale and intensive cultivation of cells. We also describe experiments which suggest that bcl-2 can reduce the specific nutrient consumption rate of cells.

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light.

Production of α-amylase by Bacillus amyloliquefaciens in batch and continuous culture using a defined synthetic medium

SummaryUsing a totally defined synthetic medium the effect of lactose and nitrogen on cell physiology and α-amylase production by Bacillus amyloliquefaciens B155 were investigated. Results showed cell growth and α-amylase production patterns to be similar regardless of the limiting nutrient and suggested stationary phase gene control of α-amylase production as opposed to a direct response to nutrient limitation.

Instability of α-amylase production and morphological variation in continuous culture of Bacillus amyloliquefaciens is associated with plasmid loss

Continuous cultivation of Bacillus amyloliquefaciens B155 using a defined synthetic medium, which supports good batch cell growth and α-amylase production, was limited by lactose, nitrogen, phosphorus and oxygen. In each case, the organism changed to a non-α-amylase-producing variant with morphological characteristics different from the original culture. Reinitiation of α-amylase production could not be achieved by plating the variant culture on solid complex medium. α-Amylase-producing and non-α-amylase-producing colonies, isolated from nitrogen-limited continuous culture were tested for plasmid DNA. The α-amylase-producing cells contained plasmid DNA, whereas non-α-amylase-producing cells did not. This suggests that plasmid structural instability (resulting in plasmid DNA loss or plasmid copy number reduction by metabolic control) accompanies loss of α-amylase production by B. amyloliquefaciens during continuous fermentations.

A study of uninfected and baculovirus-infected Spodoptera frugiperda cells in T- and spinner flasks

SummaryInsect cells have been propagated in monolayers in T-flasks or in suspension culture in spinner flasks, the latter being conducted over a range of spinner speeds. In both configurations, the cells were also infected with either wild or recombinant β-galactosidase baculovirus at MOI of 0.1, 1 and 10. The strength of both uninfected and infected cells was also measured by a micro-manipulation technique. No significant difference in growth rate was obtained between monolayer culture and suspension culture at the spinner rate which was optimum for growth. This optimum was quite sharp. At the lowest speeds cells settled, whilst above the optimum speed the spinner action led to significant cell damage. The maximum infectivity was obtained at this optimum speed which also gave maximum survival after infection. There were significant changes of cell survival and infection, even over relatively small changes of speed, and presumably energy dissipation rate. As changes in growth in turbine-agitated bioreactors have been shown to be much less, even when the energy inputs varied by two orders of magnitude, these findings throw doubt on the usefulness of spinner flasks for assessing “shear” sensitivity of cell lines. The percentage of infected cells and β-galactosidase production were significantly lower in the monolayer culture compared to that in the suspension culture at MOI values below 10 pfu/cell. This difference is explained as being due to the reduced movement of released virus particles from infected to non-infected cells in the T-flasks.

CELL DEATH BY NECROSIS AND APOPTOSIS DURING THE CULTURE OF COMMERCIALLY IMPORTANT CELL LINES

Abstract The levels of necrosis and apoptosis were quantified in CHO, SF9 insect cells, and murine plasmacytoma and hybridoma cells by acridine orange staining and fluorescence microscopy. This technique enabled the visualisation of a classic morphological feature of apoptotic cells, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was also employed to show an additional characteristic of the process, the endonuclease mediated fragmentation of DNA into multiples of 180 base pairs. Necrosis was found to be the dominant mechanism of cell death in the cultures of CHO and SF9. However, significant levels of apoptosis were found in the plasmacytoma and hybridoma cell lines. Around 81% of dead hybridoma cells were apoptotic by day 5 of a batch culture. By day 4 of the plasmacytoma batch culture the level of apoptosis was approximately 60% of dead cells.