Add the cell suspension to a round culture flask and then place the flask on a rotating rack and turn it slowly.

Operation method

Program 26.3 Rotary Bottle Culture

Principle

Add the cell suspension to a round culture flask and then place the flask on a rotating rack and turn it slowly.

Materials and Instruments

D-PBSA 0.25% natural trypsin Monolayer cells Carbon dioxide
Cultures and culture fluid containers Rotating culture bottles Hematocrit plates or electronic cell counters and fluids for counting Rotating racks

Move

1. Digest cells with trypsin and grow at usual density.

The gas phase in the rotating flask is large, so with media based on Hanks salt and air gas phase, it may be necessary to fill the flask with a small amount of 5% _CO2 (e.g., 10 L/min, 2 s). If the culture medium is buffered with _CO2/HCO3, the gas phase should be adjusted with 5% _CO2 (20 L/min, 30s~1 min, depending on the size of the flask).

2. Place the flasks on a rotating rack and rotate them at 20 rph until the cells are adherent to the wall (24~48 h).

3. When the cell density increases, accelerate the rotating speed to 60~80 rph.

4. When adding culture solution or collecting culture medium, add a small amount of 5% CO2 into the flask. When adding or collecting culture fluid, take the flask to a sterile workspace, drain the culture fluid as usual, and replace with fresh culture fluid. If volume is not a critical issue, a transfusion device or culture fluid bag is useful for adding fresh culture fluid. If the volume of culture medium is critical, add it dropwise with a pipette or measure it with a peristaltic pump.

5. Harvest the cells:

(a) Discard the culture medium, soak the cells with 50~100 ml D-PBSA, and then discard the D-PBSA.

(b) Add 50~100 ml of trypsin at 4℃. Rotate the culture flask at 20 rpm for 15 s by hand or on a rotating rack.

(c) Aspirate the trypsin and digest the cells in the culture flask for 5~15 min, then add the culture solution.

(d) Shake and rotate the culture flask and wash out the cells with a dropper.

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