CHO Transient Expression
Cells: suspension-adapted CHO K1 (ATCC #CCL-61) cells; Freestyle CHO-S cells
Yield: ~160 ± 20 mg/L of therapeutic antibody 10 days after transfection; recombinant protein: yeild similar to HEK293-FS cells.
Plasmid DNA: heavy-chain plasmid / light-chain plasmid / an EGFP plasmid ratio = 45:45:10.
Media:
- Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS) from Thermo Scientific HyClone.
- Gibco Freestyle CHO media supplemented with 8-mM glutamine + 0.1% Gibco PF-68.
Adaption: adapte the cells to a chemically defined medium from Invitrogen by weaning out serum-containing DMEM/F12 in multiple steps until our CHO-KS cells started to show good growth characteristics.
Cell culture: maintain culture in 125-mL Erlenmeyer flasks from Corning with 30-mL working volumes on a shaking platform at 125 rpm. pass cells three to four times per week so that maintenance cultures would have densities in the range of 0.2–2.0 million cells per milliliter of medium.
Transient Transfection:
One day before transfection, seed 0.5–0.75 million CHO-KS cells/mL with >96% viability. On the day of transfection, cell density is usually 1.0–1.3 million cells/mL. Use PEImax (from Polyplus Sciences) or Freestyle Max (from Invitrogen) transfection reagents, with 1 µg of DNA/mL of culture to be transfected. Ratio of DNA to transfection reagent is 1:1 (w/v) for Freestyle Max and 1:6 (w/w) for PEImax.
On the day of transfection, dilute the required amount of DNA and transfection reagents in 5% of the culture volume of OptiPRO SFM medium from Invitrogen. Mix together the content of both tubes and incubate the mixture for 10 minutes at room temperature. Add DNA complex dropwise to cells cultured in CO2 incubators set at 37 °C on a 125-rpm shaking platform. Four hours later, add in-house nutrient feed to the transfected cells and put them in an incubator maintained at 32 °C.
No centrifugation step is required
Freestyle CHO-S has higher transfection efficiency (~50%) than does CHO-KS (~20%)