For the following, prewarmed selective medium must be used in order to maintain the transfection vector in NS0 host cells. All incubations take place in a standard (37°C, 5% CO2) humidified incubator for cell culture.
1. Grow highest-producing clone to confluency in a T75 flask with selective medium.
2. Expand cells to a T225 flask until confluent (4-5 d).
3. Add entire T225 culture into an autoclaved 3-L spinner flask, followed by 1 L of selective medium. Incubate 3-4 d with continuous stirring.
4. Bring culture volume to 3 L with selective medium, incubate another 3 d.
5. When concentration reaches 8 X 105 cells/mL, aerate medium via an autoclaved sparger assembly connected to an air pump. (Use HEPA-CAP filter to sterilize input air.) Incubate another 2-3 d.
6. Add 30 mL of 100X nonessential amino acids, 60 mL of 50X RPMI essential amino acids, 60 mL of GSEM supplement, and 30 mL of 100X glucose solution. Incubate aerated culture another 3 d.
7. Pellet cells by centrifugation: 10 min at 300g in a tabletop centrifuge at 4°C.
8. Filter supernatants through a 0.2-pm bottle-top filter unit into 1-L sterile bottles. The resulting filtered supernatant is ready for purification.
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