@WildImmunity CHO serum free protocol

MaterialsComplete RPMI 5% (cRF-5) 0.5 L:

5% FBS (heat inactivated at 56C for 45 minutes)2-4 mM L-glutamine (5 ml)100 U/ml penicillin-streptomycin (5 ml)10 mM HEPES (5 ml)50 uM 2-ME (0.5 ml)1 mM sodium pyruvate (5 ml)1% non-essential amino acids (5 ml)

CHO Ex-Cell chemically defined media (cXL) (Sigma # 14361C) 1.0 L:8 mM L-glutamine (40 ml)100 U/ml penicillin-streptomycin (10 ml)10 mM HEPES (10 ml)50 uM 2-ME (1 ml)1 mM sodium pyruvate (10 ml)1% non-essential amino acids (10 ml)

Phosphate buffered salineTrypsin

Method

1. Maintain adherent CHO cell culture with appropriate selection agent (i.e. 100 μg/ml hygomycin B - Sigma # H0654-1G)

2. Seed 1-3 T175 flasks in cRF-5 (4 parts) and CHO Ex-Cell CDM (1 part) and allow cells to reach 40-80 % confluence

a. Make sure cells are in log growth phase before transfer.3. Tip out media and rinse with 10ml of sterile PBS4. Add 5 ml of trypsin and incubate at 37 °C for 5 minutes or until all cells appear

round and/or floating.5. Dilute with 15 ml of cRF-56. Thoroughly re-suspend cells using a 10 ml pipet and take an aliquot for counting7. Spin 100 g for 5 minutes at 22 °C whilst counting8. Re-suspend the cells to 1x106 in cXL9. Seed the cells into the pre-warmed, sterile impeller flask

a. Best to seed at least 50 ml, but smaller amount may be ok with small impeller flask

b. The key is having healthy, rapidly proliferating cells and seeding at a high initial density

10. Incubate the cells at 75 rpm (max)11. Count cells daily and maintain density of 5x105 to 1x106 in cXL initially.

a. Can use lower densities after cells have been fully adaptedb. Cells should remain near 100% viability

12. Supernatants may have to be removed and centrifuge if proteins have short half-lives. Otherwise continue culturing for about a week and media should need to be doubled daily.

@WildImmunity CHO serum free protocol