Do we need gradient domestication to change the brand of fetal bovine serum? How to operate it specifically

The differences between new and old serum brands mainly come from complement, hormones, lipids, and endogenous proteins. 293T cells can often be switched directly, but for MSC it is not recommended to go to 100% right away. You can first pretreat the domestic serum: after thawing, inactivate complement at 56°C for 30 min, filter through 0.22 μm, then transition with a 50% mixture for 2 passages; once the state is stable, proceed to 100%, and if unstable, return to 50% for one more passage. In addition to adherence and doubling, I also look at cell roundness, vacuolation rate, and colony formation. For MSC, focus on detecting CD105/CD73/CD90 positive, CD34/CD45/CD11b negative, and add qPCR to check SOX2/OCT4 or osteogenic differentiation markers; do not rely only on surface markers. Prepare to keep a bottle of old serum as a control, so it is convenient to revert when there is a tendency toward differentiation.

Gradient acclimation is recommended, especially since MSCs are more sensitive to changes in growth factors/hormones in serum than 293T cells. I usually start with 25% new serum + 75% old serum, observing confluence, adherence, and cell refractility every 2 passages, then proceed to 50%, 75%, and 100% once stable. Do not rely solely on passage number; consider the cells stable only when their state has stabilized. For 293T cells, the process can be appropriately shortened, but for MSCs, maintain at least 2-3 passages per gradient. In addition to adherence and doubling time, it is recommended to include trypan blue viability, apoptosis, or cell debris percentage after one day. For MSC surface markers, flow cytometry can be used to measure CD73/CD90/CD105 positivity and CD34/CD45/HLA-DR negativity. If conditions permit, supplement with osteogenic/adipogenic induction staining to avoid judging differentiation based on morphology alone.

I prefer to first use parallel experiments to decide whether acclimation is needed. The same batch of MSC/293T is divided into two groups: one directly switched to 100% domestic serum, and the other transitioned through 25%, 50%, and 75%, with 3 passages each. If the direct-switch group shows no obvious changes in doubling time, morphology, or apoptosis, it indicates that this batch has good compatibility and the acclimation period can be shortened; once filamentous stretching, vacuolation, or prolonged doubling time appears, revert to the 50% mixture and extend each gradient by 1-2 passages. During observation, add CFSE/EdU to assess proliferation, use flow cytometry to check MSC positivity for CD44, CD73, CD90, and CD105 and negativity for CD31, CD34, and CD45, and if necessary perform osteogenic/adipogenic induction to confirm that stemness has not drifted. For 293T, transfection efficiency or viral packaging titer can be added as endpoints.

We generally transition with a 1:1 ratio of old to new serum for two passages, and the cells adapt much better.

Adherent cells are best transitioned gradually, while direct replacement is generally fine for suspension cells.