Is there a feasible plan to gradually replace FBS with serum substitutes for cell domestication?

When selecting alternatives, don't focus only on price; the key is whether they support adherent cells and transfection. We typically set up three groups: 10% FBS, 5% FBS + alternative, and 2% FBS + alternative, and measure GFP transfection positive rate, target protein expression, and doubling time respectively. Adding the alternative alone is sometimes insufficient; you can supplement with insulin-transferrin-selenium, lipids, and glutamine. Under low-serum conditions, antibiotic concentrations must also be re-calibrated and cannot be copied directly.

Cutting serum to save costs requires looking at the total picture: adaptation takes time, validation consumes reagents, and it may affect the window. Low-serum banks can be established for HeLa and A549, but this is not recommended for sensitive primary cells. On the day of transfection, it is advisable to switch back to medium containing 5% FBS or 10% FBS, because transfection complexes and low-serum systems are sometimes incompatible—transfection efficiency does not automatically remain stable after adaptation. For protein expression, preliminary western/ELISA experiments should be performed for the target protein; do not judge solely by how fast the cells grow.

HeLa and A549 are relatively suitable for low serum domestication, but they should not be further reduced. Suggest starting from 10% FBS to 7.5%, stabilizing 2-3 generations, then to 5%, stabilizing 3-5 generations, and finally 2%. Record the doubling time and activity rate for each generation, and if the doubling time increases by more than 20%, switch back to half gear. During domestication, the morphology may become elongated and the edges may become brighter, and the activity rate can continue to be normal. The same batch of serum and substitute should be fixed to avoid two variables changing simultaneously, otherwise it is difficult to attribute the problem.

The domestication cycle is calculated based on at least 3 generations of each concentration, a survival rate>90%, and a doubling time recovery. Even if it is successful at 10% to 2%, it will take 6-10 generations or more. Newly domesticated cells should not be subjected to critical experiments, but should be stabilized for at least three more generations and validated through cryopreservation and recovery. During the process, there are a large number of loose adhesion, air bubbles, and multiple nuclei, which are mostly due to the rapid descent or unsuitable substitutes. It is recommended to retreat 10% and let it rest for two weeks. Domesticated cells should be kept in multiple frozen batches to prevent subsequent aging.