Transferring CHO Cells From Monolayer to Suspension Culture

1. 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.
2. 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.
3. 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.
4. Decant the supernatant and repeat step 3.
5. Decant the supernatant and resuspend the cells in 5 ml of medium. Remove an aliquot to determine viable cell count.
6. 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%.
7. Loosen caps for gas exchange and place at 37^oC 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.

8. Check viable cell density daily to establish growth kinetics in suspension culture.
9. 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).
10. 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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