Editing HEK293-EBNA1 (section)

=== METHOD  ===

Conduct all operations aseptically in a laminar flow hood. Cells growing in serum-free media are highly sensitive to chemical contamination; thorough rinsing of all equipment that contacts the cells with highly pure water is compulsory.<br>Cell Thawing and Maintenance 

1. Prepare a 15-mL conical tube containing 10 mL of prewarmed cell culture medium appropriate for the cell line to be used. 

2. Thaw the cells rapidly in a 37°C water bath. 

3. Add the cells to the 15-mL tube. Invert to mix. 

4. Dilute a 100-μL aliquot of cells with 100 μL of PBS containing 25 mg/mL erythrosin B. Determine the cell density and viability using a hemocytometer. 

5. Centrifuge the cells at 200g for 5 min at room temperature. 

6. Decant the supernatant. Loosen the pellet by gently tapping the tube. Dilute the cells to 2.5 × 105 cells/mL in a 125-mL disposable shake flask using cell culture medium. 

Operating culture volumes must be in the range of 16%-33% of the nominal shake flask volume for sufficient oxygenation. 

7. Place the shake flask on the orbital shaker (~120 rpm) in the incubator. 

Center the shaker in the incubator. Make sure that the walls, door, and wire are not contacted during agitation. 

8. Count the cells at 48 h post-thawing. If necessary, dilute to 2.5 × 105 cells/mL using cell culture medium. 

9. When the doubling time is 24 h for two consecutive passages, subculture every 2 or 3 d to maintain cell densities between 2.5 × 105 and 1.2 × 106 cells/mL using medium supplemented with G418 (25 μg/mL or 50 μg/mL, for 293-6E or 293E cells, respectively). 

See Troubleshooting. 

10. Dilute the cultures to 1.5 × 105 or 7.5 × 104 cells/mL for weekends or long weekends (3 or 4 d, respectively). 

Cell Freezing 

11. Prepare the freezing mixture by adding DMSO to fresh cell culture medium (10:90, v/v). 

12. Label the cryovials. Place in a small Styrofoam box. Prechill the large Styrofoam box in a -80°C freezer. 

13. Count the cells. Determine the volume needed for cryopreservation. 

Cells must be in the exponential phase (between 8 × 105 and 1.2 × 106 cells/mL). 

14. Centrifuge the cells at 200g for 5 min. Decant the supernatant. Dissociate the pellet by gently tapping the tube. 

15. Add the freezing medium drop-wise to the cells while swirling the tube to obtain the desired density (i.e., 5 × 106 to 5 × 107 cells/mL per vial). 

16. Quickly aliquot the cells into the labeled vials. Immediately transfer to a -80°C freezer. Do not open the freezer door for at least 2 h. 

Alternatively, freeze cells at 1°C/min in a programmable cooler. 

17. Transfer the vials to liquid nitrogen freezer storage (vapor phase) the following day. 

Store for a maximum of 4 d. 

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