Editing HEK293-EBNA1 (section)

=== TROUBLESHOOTING  ===

Problem: The cells do not grow well. 

[Step 9] 

Solution: If cells do not grow with the expected doubling time, consider the following: 

1. The cultureware is not sufficiently clean. Glassware for all culture manipulations and reagent preparations must be rinsed at least three times with high-purity H2O. 

2. The H2O used may not be sufficiently pure. Check the source of the ultrapure H2O. 

3. Check that the filtration system (if applicable) used to make the ultrapure H2O is functioning properly. 

4. Cell aggregation can give a false impression of slower growth rates. Check regularly that the orbital shaker is at the right speed and that the incubator is at the correct temperature and is supplying the prescribed amount of CO2. 

5. The cells may be too old. The number of cell passages indicates the age of the cells in service. A new cryovial can be thawed every 3 mo, on average. 

6. Verify expiration dates, culture medium lots, and batches of additives used (e.g., Pluronic F-68 and antibiotics). 

7. Check for mycoplasma contamination; if positive, thaw new cells<br> 

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293E cells were maintained in low-calcium hybridoma serum-free medium (LC-SFM) supplemented with 1% BCS and 50 μg/mL G418. Cells were maintained in agitated (110-130 rpm) shake flasks under standard humidi- fied conditions (37 °C and 5% CO2). One day prior to transfection, cells were diluted at a density of 0.25 × 106/ mL in LC-SMF growth medium, supplemented with 1% BCS and transfected 18-24 h later when the density reached approximately 0.5 × 106 cells/mL. Plasmid DNA and PEI were mixed together in LC-SFM medium at 1 and 2 μg/mL of culture to be transfected, respectively. Upon the addition of PEI to the DNA, the mixture was vortexed, incubated for 15 min, and added to the cells. Cells were harvested 72- 100 h post-transfection (hpt) by centrifugation at 1500g for 10 min, followed by a wash with ice-cold PBS and a subsequent centrifugation step at 1500g for 15 min. The cell pellets were frozen at -80 °C.<br><br> 

Yves Durocher (NRC Montreal) compared PEI based transient transfection in 293EBNA and CHO cells. (1) The 293EBNA clone 6E in combination with the pTT5 vector (4.4 kb) yields 150 to 250 mg/L of mAb at 6 days post-transfection (+/– oriP has a 3-4 fold effect on expression). (2) Data on a newly generated CHO-EBNA1 cell clone (3E7) was also presented. EBNA1 expression was stable w/o selection pressure over 4 months. GFP expression levels for CHO 3E7 and “normal” CHO using a pTT-GFP vector were compared. Expression levels were increased 4-5 fold. Similar improvements in titer were observed for secreted alkaline phosphatase (SEAP). mAb expression levels in CHO-3E7 reached 150 mg/L within 12 days. <br> 

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Expression constructs were tested in deep, 24-well plates using a slight variation of standard techniques.14 2936E Human Embryonic Kidney (HEK) cells stably expressing EBNA1 (NRCC) were cultured in suspension using 293 Freestyle media sup- plemented by 25 lg/mL Geneticin and 0.1% solution of Pluronic F-68 (all cell culture reagents from Invitrogen). Transfection complexes were generated in the 24-well plates (Seahorse Labware) by adding 1.2 lg of DNA and 8.4 lg of 25 kDa linear polyethy- leneiminie (PEI) from (Polysciences) in 200 lL Free- style media. DNA: PEI was at a 1:7 ratio with 0.25 lg DNA/mL of culture. The DNA/PEI mixture was allowed to incubate for 10 minutes at room tempera- ture. Cells were directly dispensed to the 24-well plate at 5 mL/well and 1 Â 106 cells/mL. Plates were shaken overnight at 37o C and 5% CO2 on a Titramax 101 shaker platform (Heidolph). Twenty-four hours following the transfection the wells received a peptone feed of 0.5% TN-1. Plates were replaced on the shakers in the incubator and harvested 5 days post transfection. <br>

[[Category:293_Expression]] [[Category:Protocols]]