42 Large-Scale Cell Culture
391
- Magnetic stirrers and magnetic bars
- If the magnetic bar rotates on the bottom of the flask, the cells may
be crushed by rolling into the interstices between the bottom surface and the magnet; therefore, it is preferable to use a flask that has
a magnetic bar suspended from the top of the flask
- Culture medium
Procedure
l. Harvest cells from a stock culture.
2. Place the cells into spinner flasks.
3. Run the magnetic stirrer at an appropriate speed (the optimum speed
is dependent on the cell line and composition of the medium used).
Results
Reuveny et al. (1993) cultured Spodoptera frugiperda cells in a 12S-ml
flask with 50 ml ICSF-WB serum-free medium at a rate of stirring of
140 r.p.m. Temperature affected cell growth and the maximum cell concentration was obtained at 27°C after 120 h. Shuler et al. (1989) cultured
T. ni (TN-368) in a SO-ml spinner flask to determine the optimal stirring
rate. A lower stirring rate (30 r.p.m.) did not maintain cells in suspension, and resulted in cell aggregation. At higher speed (120 r.p.m.), an
extensive amount of cell lysis occurred. Good cell growth was obtained
at a stirring rate of 60 r.p.m., and cell density increased from l.2 x 10 5 to
l.0 x 106 cells/ml. Hensler et al. (1994) cultured SF-9 cells in a 2S0-ml
spinner flask fitted with a DO and pH probe. In Hink's TNM-FH medium supplemented with 5% heat-treated FBS and 0.1 % Pluronic F-68 at
28°C and 65 r. p.m., Hensler et al. (1994) found that the cell density reached
5 X 10 6 cells/ml.
Subprotocol 4
Stirred Tank Bioreactor Culture
This system is applicable for cell suspensions of between 3 and 150 Lit
has generally been considered that the shear force generated by the stirring apparatus is the main cause of cell damage; however, it has become
evident that the shear force generated by an air bubble is considerably
more detrimental. To overcome this drawback, bubble-free aeration techniques in stirred tanks, which can be fitted with semipermeable membrane tubing for oxygenation, have been developed.
Solutions
391
- Magnetic stirrers and magnetic bars
- If the magnetic bar rotates on the bottom of the flask, the cells may
be crushed by rolling into the interstices between the bottom surface and the magnet; therefore, it is preferable to use a flask that has
a magnetic bar suspended from the top of the flask
- Culture medium
Procedure
l. Harvest cells from a stock culture.
2. Place the cells into spinner flasks.
3. Run the magnetic stirrer at an appropriate speed (the optimum speed
is dependent on the cell line and composition of the medium used).
Results
Reuveny et al. (1993) cultured Spodoptera frugiperda cells in a 12S-ml
flask with 50 ml ICSF-WB serum-free medium at a rate of stirring of
140 r.p.m. Temperature affected cell growth and the maximum cell concentration was obtained at 27°C after 120 h. Shuler et al. (1989) cultured
T. ni (TN-368) in a SO-ml spinner flask to determine the optimal stirring
rate. A lower stirring rate (30 r.p.m.) did not maintain cells in suspension, and resulted in cell aggregation. At higher speed (120 r.p.m.), an
extensive amount of cell lysis occurred. Good cell growth was obtained
at a stirring rate of 60 r.p.m., and cell density increased from l.2 x 10 5 to
l.0 x 106 cells/ml. Hensler et al. (1994) cultured SF-9 cells in a 2S0-ml
spinner flask fitted with a DO and pH probe. In Hink's TNM-FH medium supplemented with 5% heat-treated FBS and 0.1 % Pluronic F-68 at
28°C and 65 r. p.m., Hensler et al. (1994) found that the cell density reached
5 X 10 6 cells/ml.
Subprotocol 4
Stirred Tank Bioreactor Culture
This system is applicable for cell suspensions of between 3 and 150 Lit
has generally been considered that the shear force generated by the stirring apparatus is the main cause of cell damage; however, it has become
evident that the shear force generated by an air bubble is considerably
more detrimental. To overcome this drawback, bubble-free aeration techniques in stirred tanks, which can be fitted with semipermeable membrane tubing for oxygenation, have been developed.
Solutions