- Decant medium from a T-25 flask and wash monolayer with 3 ml (5 ml for a T-75 flask) of Calcium and Magnesium-free D-PBS, Cat. No. 14190. Culture should be 50 to 80% confluent to ensure that the cells are actively dividing.
- Decant PBS. Add 3 ml (5 ml for a T-75 flask) of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA·4Na, Cat. No. 25300). Swirl briefly and decant all but enough trypsin-EDTA to leave a thin film of liquid over the monolayer. Observe cells under microscope. When cells have rounded up, rap flask on bench top to dislodge the cells.
- Add 6 ml of medium (10 ml for a T-75 flask) to the cell suspension and centrifuge at 100 X g for 4 min. The diluted trypsin will be aspirated off with the supernatant. For serum-free medium, we recommend using, soybean trypsin inhibitor (cat. no. 17075) stop the enzymatic action. Make a 0.25 mg/ml stock solution of soybean trypsin inhibitor. Use this stock solution 1:1 (v:v) with trypsin solution.
- Decant the supernatant and repeat step 3.
- Decant the supernatant and resuspend the cells in 5 ml of medium. Remove an aliquot to determine viable cell count.
- Determine volume of cell suspension needed for 5 x 105 viable cells/ml. Add an appropriate volume of medium and cell suspension to a sterile spinner or shake flask. Leave sufficient headspace for adequate gas exchange (i.e., 100 ml of medium in 250-ml spinner flask or 75 to 100 ml of medium in a 250-ml shake flask). For serum-free medium that does not already contain a shear protectant, add PLURONIC® F68 to a final concentration of 0.1%.
- Loosen caps for gas exchange and place at 37oC in a humidified atmosphere of 5 to 10% CO2 in air. Set impeller speed to 75-95 rpm (for Corning Spinners; for paddle-type impellers, speed may need to be decreased). Shake flasks can be rotated on an orbital shaker platform at 125 to 135 rpm.
Note: You can set spinner flask impeller speed to your normal speed (80 to 100 rpm). If the cell viability drops below 85%, reduce the speed in 10 rpm decrements until the viability is good.
- Check viable cell density daily to establish growth kinetics in suspension culture.
- When viable cell density reaches 1 x 106 cells/ml (or on day 4 post-planting). Passage cells at 5 x 105 cells/ml (centrifugation followed by fresh medium may be necessary if the cell density has not reached 1 x 106 cells/ml by day 4).
- Once cell density reaches at least 1 x 106 cells/ml with a viability of at least 90% by day 3 post-planting for 3 passages, the cells may be considered adapted to suspension culture. Seeding density can then be reduced to 2 x 105 to 3 x 105 cells/ml.
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