Editing 293EF Protein Production (section)

== <br>Subculture Expi293F Cells <br>  ==

When the cells reach a density of approximately 3 × 10^6– 5 × 10^6 viable cells/mL, typically every 3–4 days, split the Expi293 F culture to 0.3 × 10^6–0.5 × 10^6 cells. When maintaining Expi293F Cells.&nbsp; Usually a 125-mL or 250-mL polycarbonate, disposable, sterile Erlenmeyer flask is used for 25–40 mL or 50–80 mL total working volume of cell suspension, respectively. 

Glass flasks without baffles may be used, but thorough cleaning after each use is essential to avoid potential toxicity, which is more problematic in serum-free cultures. 

1. Determine viable and total cell counts 

2. Using the cell density determined in Step 1, calculate the split ratio needed to seed the new shaker flask at 0.3 × 10^6–0.5 × 10^6 viable cells/mL. 

3. Dilute the cells in fresh, pre-warmed Expi293TM Expression Medium to give a final cell density of 0.3 × 10^6–0.5 × 10^6 viable cells/mL in the desired final volume. 

4. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 125 rpm. 

5. Repeat Steps 1–4 as necessary to maintain or expand cells. 

=== Scale Up<br>  ===

It is possible to scale up the Expi293 F cultures in spinner flasks or bioreactors. The appropriate spinner or impeller speed and seeding density should be determined and optimized for each system. At Life Technologies, we have determined the optimum spinner speed to be 100–130 rpm, and impeller speed in Celligen® stirred tank bioreactors to be 70–100 rpm. We recommend seeding the cells at 0.3 × 10^6–0.5 × 10^6 viable cells/mL.<br><br>