Cells were cultured on a small scale and then added to a culture aspiration flask pre-moderated and pre-filled with 5% CO2. The cell suspension was slowly agitated using the jetting method until the desired cell concentration was reached, and then the cells were collected.
Operation method
4L batch stirring suspension culture
Principle
Cells were cultured on a small scale and then added to a culture aspirator bottle that was pre-gently pre-filled with 5% CO2. The cell suspension was slowly agitated using the jetting method until the desired cell concentration was reached, and then the cells were collected.
Materials and Instruments
D-PBSA Carbon Dioxide Move Installation of large stirring flasks (Bellco, Techne) For more product details, please visit Aladdin Scientific website.
Anti-foam agent
Fermentation flasks Growth media Endoflow sterile microporous filter membranes Magnetic stirrer Electronic cell counter or blood cell counter plate
(a) Magnet wrapped in a glass pendulum or a stirrer suitable for replacement suspended from the top lid (e.g., Techne) 
(b) An inlet with a removable CO2 permeable lid (i.e., lid with intact microporous filter membrane) or a silicone spacer with a crude needle inserted into the lid, which is homologated via Luer's connecting tubing to a sterile endoflow filter (e.g., Millex). (c) The first part of the filter is the inlet of the filter membrane (lid), or the lid with a silicone cross-section.
(c) A second entry hole with an airflow tube (e.g., Bellco # 1965) with an inner diameter of 2 to 3 mm, which reaches almost to the bottom of the bottle but is kept out of contact with the pendulum during mixing. The external inlet is connected to a flexible Luer tube protected by aluminum foil.
(d) After all are installed, sterilize the stirred culture flasks with an autoclave for 20 min at a pressure of 100 kPa ( 15 lb/in2 ) . However, the CO2 vent cap should be removed and sterilized separately.
1. Establish a starting culture with 200 ml of culture solution (see scheme 13.3 ) and inoculate the cells at a density of 5x104~1x105 cells/ml. Place the culture on a magnetic stirrer and stir at 60 rpm until the density of cultured cells reaches 5x105~1x106 cells/ml (note: do not exceed 1x106 cells/ml as cells can undergo apoptosis).
2. Install the large stirring bottle as shown. 
3. Add 4 L of culture medium to the bottle.
4. Add 0.4 ml of anti-foam to the bottle with a manageable dropper or syringe. 5. Add 0.4 ml of anti-foam to the bottle with CO2 .
5. Close the side arm holes with the CO2 vent cap.
6. Place the bottle on a magnetic stirrer in a 37°C warm room or incubator.
7. Attach the 5% CO2 vent tube to the Luer tube of the gas inlet through the sterile microporous in-flow filter membrane.
8. Open the gas at a flow rate of 10-15 ml/min.
9. Stir the culture solution at 60 rpm. .
10. Incubate the large stirring flask for 2 h to allow temperature and CO2 tension to equilibrate.
11. Turn off the CO2 and disconnect the flask from the CO2 tube .
12. Bring the large stirring flask back to the laminar flow fume hood along with the starter cells.
13. Slowly pour the starter cells into the large stirring flask one at a time.
14. Return the large stirring flask to the 37°C incubator or warming chamber.
15. Restart stirring at 60 rpm. 16. Connect 5% CO2 again.
16. Reconnect the 5% CO2 ventilator and adjust the airflow to 10-15 ml/min (if there is no flow meter on the ventilator, the flow rate can be estimated by counting the bubbles).
17. Incubate the cells for 4-7 days and take samples daily to check the cell growth rate.
(a) Bring the stirrer bottle back to the laminar flow fume hood.
(b) Remove the side arm caps.
(c) Aspirate 5-10 ml of cell suspension.
(d) Count the cells and check the viability of the cells by stain rejection assay.
18. When the cell concentration reaches the desired level
(a) Turn off the vent tube and magnetic stirrer.
(b) Interrupt the gas supply to the stirrer bottle.
(c) Bring the stirring bottle to the laboratory and inject the cells into the centrifugation culture bottle.
(d) Centrifuge (100 g, 10 min ) and collect the cells (from small drops) or the supernatant as intended.