Editing 293EF Protein Production (section)

=== Thawing<br><br>  ===

1. Remove the vial of cells from liquid nitrogen and place it in a 37°C water bath for 1 to 2 minutes to thaw the cells rapidly with gentle agitation. Do not submerge the vial in the water. 

2. Just before the cells are completely thawed, decontaminate the vial by wiping it with 70% ethanol before opening it in a Class II biological safety cabinet 

3. Using a 2-mL or 5-mL pipette, transfer the entire contents of the cryovial into a 125-mL polycarbonate, disposable, sterile, vent-cap Erlenmeyer shaker flask containing 29 mL of pre-warmed Expi293TM Expression Medium. 

4. Immediately post-thaw, monitor viable cell density and viability using a hemocytometer with the trypan blue exclusion method or using an automated cell counter. Cell density should be approximately 0.3 × 10^6 cells/mL and cell viability should be above 80%. 

5. Incubate the cells in a 37°C incubator with humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 125 rpm. 

6. The day after thawing, determine the viable and total cell counts (see protocol on page 6). Cells viability will drop slightly but should remain above 70% and reach over 90% by day 2–4 post-thaw. Thaw out a new vial of Expi293F Cells if the cell viability is below 70%. 

7. Once the culture has reached &gt;1 × 10^6 viable cells/mL (typically 2–4 days), subculture the Expi293 F Cells by seeding shaker flasks at 3 × 10^5 viable cells/mL in pre-warmed Expi293 Expression Medium. Use&nbsp; a 125-mL or 250-mL polycarbonate, disposable, sterile, Erlenmeyer flasks for 30 mL or 60 mL total working volume of cell suspension, respectively. 

8. Continue to monitor cell density and viability. Once the culture has reached &gt;3 × 10^6 viable cells/mL (typically 3–4 days), subculture the Expi293F Cells by seeding shaker flasks at 3 × 10^5 viable cells/mL in pre-warmed Expi293 Expression Medium.