Does the direct switch of CHO cells from serum-containing to serum-free medium require an adaptation step?

Some CHO serum-free systems do not contain L-glutamine or require the separate addition of HT supplement, so the formulation should be checked before switching. If it is a CD medium, it is usually necessary to add the manufacturer's matching feed, and attention should be paid to osmolality and trace elements. We encountered a severe drop in viability after a direct switch, and ultimately found that the medium lacked glutamine; it recovered after supplementation. During the serum-free phase, the medium exchange frequency can be maintained at 2-3 days, depending on density and pH.

If the cells are in good condition and in the logarithmic phase, you can switch directly, but increase the inoculation density to 1.5-2×10^6/ml. When changing the medium, centrifuge the old medium and resuspend the cells in serum-free medium. After direct switching, some cells will die in the first 48 hours, and viability may drop to around 70%. As long as not too many die, they can usually adapt within 5-7 days. However, commercial serum-free medium is best preheated to 37°C in advance, and glutamine or the manufacturer-recommended additives should be supplemented.

We adapted CHO-K1 from 10% FBS gradually: first reduced to 5% FBS + 5% serum-free, passaged 1-2 generations; then 2.5% FBS + 7.5% serum-free, passaged 1-2 generations; then 1% FBS + serum-free, and finally completely serum-free. At each step, we proceeded to the next step only after observing viability >85%. Passage at 1:3-1:4 every 2-3 days, and don't wait until the cells are too dense. This way the cycle takes about 10-14 days, but it's more stable than direct switching.

Whether gradual or direct, it is recommended to first keep a batch of serum-containing cryopreserved seeds or a continuous culture backup; do not switch everything over at once. After serum-free adaptation, continuously monitor viability, doubling time, and recombinant protein expression, and ensure stability for at least 3 passages before proceeding to formal production. Small-scale parallel controls can also be performed: serum-containing vs. gradual vs. direct, to assess differences in expression levels. In our case, the gradual group ultimately had the highest protein yield, while the direct group was about 20% lower in the early stage.

When gradually reducing serum, do not centrifuge and change the medium frequently. Instead, use half-volume medium exchange or directly subculture and dilute to minimize mechanical damage. Our procedure: at each passage, dilute and inoculate with serum-free medium without washing the cells, allowing residual serum to be gradually diluted out. For example, after a 1:4 subculture, the serum concentration automatically becomes one quarter. This is gentler than centrifugation-based serum removal, and the cells do not clump and adapt more quickly.