cMyc increases cell number through uncoupling of cell division from cell size in CHO cells

<p>Abstract</p> <p>Background</p> <p>Over the past decades, the increase in maximal cell numbers for the production of mammalian derived biologics has been in a large part due to the development of optimal feeding strategies. Engineering of the cell line is one of probable approaches for increasing cell numbers in bioreactor.</p> <p>Results</p> <p>We have demonstrated that the over-expression of the <it>c-myc </it>gene in immortalised CHO cells can increase proliferation rate and maximal cell density in batch culture compared to the control. The changes were attributed to a rapid transition into S-phase from a shortened duration of G₁phase and to the uncoupling of cell size from cell proliferation. To achieve the >70% increase in maximal cell density without additional supply of nutrients the cells underwent an overall reduction of 14% in size as well as a significant decrease in glucose and amino acid consumption rate. Consequently, the total biomass accumulation did not show a significant change from the control. The amount of hSEAP-hFc activity of the over expressing <it>c-myc </it>cell line was found to be within 0.7% of the control.</p> <p>Conclusion</p> <p>It is shown that the manipulation of cell cycle kinetics and indirectly cell metabolism gives higher cell densities in CHO batch cultures. The unaltered apoptotic rate supported the proposition that the increase in cell number was a result of enhance cell cycle kinetics and cellular metabolism rather than increasing viability. Production of hSEAP-hFc from a constitutive <it>c-myc </it>over-expressing cell line did not increase with the increase in cell number.</p

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day-1) for 4 days, increasing gradually to 2 day-1 at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line. © 2004 Kluwer Academic Publishers

Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells

Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d-1 to 0.2 d-1. Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d-1 and increased to 55% when the dilution rate was reduced to 0.2 d-1. As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d-1. The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d-1. By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d-1. For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 μg·ml-1 as the dilution rate was reduced from 0.8 to 0.2 d-1. With an initial increase from 2 to 15 μg·ml-1 as the dilution rate was reduced from 0.8 to 0.4 d-1, the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d-1. A good correlation between specific antibody production rate and the percentage of G2/M cells was observed. © 2005, The Society for Biotechnology