Will the antibody expression level of 293 cells drop after replacing serum with serum-free medium?

After serum-free adaptation, growth will be slower during the first 1-2 passages, so do not rush to transfect. It is recommended to perform transient transfection only after at least 3 stable passages following recovery, with a seeding density controlled at 0.5-1×10^6/ml, and to replace with fresh serum-free medium one day before transfection. When we encounter decreased expression, it is usually not the medium itself, but rather the cells not having adapted or incorrect feeding timing. For 2-3 L scale, triangular shake flasks can be used; remember to calibrate the shaker speed and CO2 to avoid volume changes from evaporation affecting the results.

Do not switch directly from 10% FBS to complete serum-free medium. Although 293 cells are robust, a direct switch can easily lead to poorer attachment, clumping, and reduced viability during transfection. We use a transition of 5% FBS:serum-free = 1:1 for one passage, then switch to 25% serum/serum-free, and finally to fully serum-free, with each step lasting about 2 days. If time is tight, at least mix serum-free medium and serum-containing medium 1:1 for 24 hours before switching to serum-free. Transfection is not recommended when viability is below 85%.

For single-batch volumes within 300 L, focus on domestic serum-free transient transfection media; some formulations are benchmarked against imported ones and support 293F/293E suspension culture. When comparing, don't look only at the list price—ask clearly whether glutamine is included, whether an anti-clumping agent must be added separately, and whether feed pricing is included. Also ask the supplier to provide reference 293 transient expression data and a COA for the trial sample lot. During selection, we tested three domestic brands in parallel and saw expression differences of up to 30%, so it is essential to obtain trial samples and run actual transfection tests.

Several domestic brands offer 50-100 ml trial sizes, some of which can be applied for free or shipped at the cost of freight, and some agents of imported brands also have trial policies. With a budget of within 300 per liter, choosing a domestic Fed-batch type or an imported basic model is generally sufficient, but additional feeding may be required during the transient expression stage. Delivery time depends on the region; commonly used domestic models are generally in stock and can arrive in 3-5 days, while imported ones may take 1-2 weeks. It is recommended to ask 1-2 agents at the same time to reserve inventory.

We have performed suspension adaptation of HEK293 and then switched from serum-containing to serum-free conditions; antibody expression did not drop, and in fact the batch-to-batch variability was much lower. However, the serum-free system is more sensitive to PEI transfection, so the plasmid DNA-to-PEI ratio needs to be re-screened, generally starting around DNA:PEI = 1:3; it is best to choose a specialized medium that supports transient transfection and add feeds according to the manufacturer's recommendations. At the 2–3 L scale, first perform evaluation in 50–100 mL shake flasks; do not go directly to larger flasks.