Membrane and microsparging aerations in long-term high-density perfusion cultures of animal cells

The profile of the inner-tubing gas pressure for a tubular membrane aeration system was quantified. The correlations among the overall volumetric oxygen transfer coefficient (k_La), the inner-tubing pressure, the tubing tightness, and the gas throughput are experimentally analyzed. A mathematical model was developed to describe the underlying phenomena. The results established the base for comparison with other aeration techniques.; A novel method employing in situ laser imaging technology to monitor bubbles and cells, and analyze bubble size distributions in a micro-sparged bioreactor was developed. The effects of bioreactor operations on bubble size distributions were determined with following results: (1) Spargers with larger pores produced larger bubbles in most cases; (2) Higher sparging rates resulted in bubble size increases up to 10%; (3) Pluronic F68 shrank bubbles up to 30%. When the concentration of Pluronic F68 exceeded 1 g/L, no additional impact was observed; (4) Emulsion silicone antifoam up to 25 ppm had no impact on bubbles; (5) Cell density (up to 22 × 106 cells/mL) or culture age has no effect on bubble sizes.; In multiple 15-L long-term high-density cultures of animal cells, the correlations between sparging rate and cell damage for using 0.5 μm and 15 μm-pore spargers were quantified. At cell density of 2 × 10 7 cells/mL, sparging above 0.025 vvm using the 0.5-μm sparger was detrimental to cells, while 0.054 vvm was detrimental for the 15-μm sparger. A model was developed to predict the rate of cell death resulted from cell-bubble interactions for high-density industrial animal cell cultures.; The effect of high superficial velocity of sparging gas on cells at the sparger surface proved insignificant.; A new dissolved CO₂ sensor proved to be reliable for long-term use in industrial perfusion cell cultures. A novel method for the control of dissolved CO₂ while simultaneously maintaining DO₂ and pH setpoints was developed. The continuous control of dissolved CO ₂, DO₂ and pH is achieved by simultaneously adjusting the total sparging rate as well as the ratio of O₂, N₂ and CO₂ gas contents. This control strategy enables optimization of dissolved CO₂ in industrial culture processes and allows for improved cell growth and protein production.