Ryoichi Haga

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×108 cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×107 cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×107 cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.

Expressions of mass transfer coefficients of bubbles and free surface of culture tanks using the k–ɛ turbulence model

For mammalian cell culture, getting a continuous supply of oxygen and extracting carbon dioxide are primary challenges even in the most modern biopharmaceutical manufacturing plants, due to the low oxygen solubility and excessive carbon dioxide accumulation. In addition, various independent flow and mass transfer characteristics in the culture tanks vessel make scale-up extremely difficult. One method for overcoming these and providing rational optimization is solving the fluid and mass transport equations by numerical simulation. To develop a simulation program, it is decisively important to know mass transfer coefficients of gaseous species in the culture tank. In this study, oxygen mass transfer coefficients are measured using a beaker with a sparger and impellers. In order to investigate the formulation of the mass transfer coefficients, the turbulent flow statistics is calculated by a CFD code for all cases, and the expressions of the mass transfer coefficients are established as functions of the statistics. Until now, the expression by Kawase is known in this field. This expression becomes a function only of energy dissipation rate ɛ. It does not coincide with the conventional experimental fact that mass transfer coefficient is proportional power 0.5 of impeller rotation speed. The new mass transfer coefficient is dependent on both of energy dissipation rate ɛ and turbulent flow energy k. It satisfies the relation of power of 0.5 of impeller rotation speed.

High-Density Animal Cell Culture by Gas Sparging

In order to enhance the oxygen supply for an animal cell culture, several foam-suppressing methods which enable direct gas sparging into culture medium were investigated. When an anti-foaming reagent was added to the medium to suppress foaming, the amount of reagent needed to be controlled in accordance with the aeration rate. With sparger optimization, increasing the sparger pore size reduced the remaining foam time. However, with the sparger pore size larger than certain value, the remaining foam time became constant, and this pore size can be regarded as the optimum. A defoaming device with a hydrophobic net has also been developed, and a culture system equipped with this defoaming net was constructed. A series of culture experiments was performed to confirm the efficacy of the defoaming net, and enhanced cell density and productivity were confirmed.