3.7 Preparation of a
Bioreactor
for the Cultivation
of Sf-9 Insect Cells
and Viral Infection
3.7.1 Setup preparation
for the Cultivation
1. Attach filters and tubing to the connector caps of medium,
inoculum, base and acid bottles (see Note 24).
2. Fill phosphoric acid and sodium hydroxide into the acid and
base bottles and close the caps (see Note 24).
3. Attach tubing clamps to all tubes.
4. Connect tubes for sampling and pH regulation to the
bioreactor.
5. Mount sterile air filters to the gas in-let and outlet.
6. Calibrate the pH probe according to the manufacturer’s
instructions.
7. Install the pH and DO probe in the bioreactor head plate at a
suitable depth.
8. Use aluminum foil to cover all probe connectors and filters.
9. Open all tubing clamps, except those for acid and base, and
autoclave the bottles and the bioreactor.
10. After autoclaving, close all tubing clamps and let the equipment cool down to room temperature.
3.7.2 Initiation
of the Cultivation
1. Count the cells in the shake flask culture (see Subheading
3.4.2).
2. Calculate the inoculum volume needed for an initial cell concentration of 1 Â 10
6 cells/mL.
3. Transfer the cultivation medium into a sterile medium bottle
while working under the clean hood.
4. Disinfect the connectors and connect the medium bottle to the
bioreactor.
5. Open the tubing clamps of the medium bottle tube and use
gravity to fill the reactor with the medium.
6. Set and start the controllers for stirrer and temperature (e.g.,
28
C, 70 rpm).
7. Start aeration with 100% air and 0.1 vvm to equilibrate the
medium with ~21% oxygen.
8. One-point calibrate the dissolved oxygen to 100% after the DO
value is stabilized, the system is mixed, and a temperature of
28
C is reached.
9. Activate acid and base pumps until the tubing is filled up to the
reactor inlet. Set pH controller to pH 6.3 and activate the pH
controller.
10. Transfer the calculated inoculum volume from the baffled
shake flask into the inoculum flask while working under the
clean hood.
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Julie Harnischfeger et al.
Bioreactor
for the Cultivation
of Sf-9 Insect Cells
and Viral Infection
3.7.1 Setup preparation
for the Cultivation
1. Attach filters and tubing to the connector caps of medium,
inoculum, base and acid bottles (see Note 24).
2. Fill phosphoric acid and sodium hydroxide into the acid and
base bottles and close the caps (see Note 24).
3. Attach tubing clamps to all tubes.
4. Connect tubes for sampling and pH regulation to the
bioreactor.
5. Mount sterile air filters to the gas in-let and outlet.
6. Calibrate the pH probe according to the manufacturer’s
instructions.
7. Install the pH and DO probe in the bioreactor head plate at a
suitable depth.
8. Use aluminum foil to cover all probe connectors and filters.
9. Open all tubing clamps, except those for acid and base, and
autoclave the bottles and the bioreactor.
10. After autoclaving, close all tubing clamps and let the equipment cool down to room temperature.
3.7.2 Initiation
of the Cultivation
1. Count the cells in the shake flask culture (see Subheading
3.4.2).
2. Calculate the inoculum volume needed for an initial cell concentration of 1 Â 10
6 cells/mL.
3. Transfer the cultivation medium into a sterile medium bottle
while working under the clean hood.
4. Disinfect the connectors and connect the medium bottle to the
bioreactor.
5. Open the tubing clamps of the medium bottle tube and use
gravity to fill the reactor with the medium.
6. Set and start the controllers for stirrer and temperature (e.g.,
28
C, 70 rpm).
7. Start aeration with 100% air and 0.1 vvm to equilibrate the
medium with ~21% oxygen.
8. One-point calibrate the dissolved oxygen to 100% after the DO
value is stabilized, the system is mixed, and a temperature of
28
C is reached.
9. Activate acid and base pumps until the tubing is filled up to the
reactor inlet. Set pH controller to pH 6.3 and activate the pH
controller.
10. Transfer the calculated inoculum volume from the baffled
shake flask into the inoculum flask while working under the
clean hood.
112
Julie Harnischfeger et al.