Must the medium be switched to exosome-depleted fetal bovine serum for extracellular vesicle isolation?

Self-made exosome-depleted serum can be prepared by ultracentrifugation: first filter regular FBS through a 0.22 μm filter to remove large particles, then centrifuge at 100,000 × g at 4°C for 16–18 hours, gently collect the supernatant without disturbing the pellet at the bottom of the tube, and finally filter-sterilize. The volume processed per run is limited by the rotor and tube capacity, it is relatively time-consuming, and strict aseptic technique is required. Self-made preparations may not completely remove all bovine-derived exosomes; it is recommended to use NTA and Western blot to detect the background of CD63, CD9, and TSG101 to verify the depletion efficiency. If ultracentrifugation conditions are available, this approach can save considerable money.

If the budget is limited, you can first try "short-term starvation + low-serum conditions" or a commercial serum-free medium. Some tumor cells can maintain exosome secretion in 1-2% FBS, and the background is also lower than with 10% serum; you can also look for domestic exosome-depleted serum or small-pack trial samples from distributors, which may be cheaper than imported ones. The key is to use NTA, BCA, and markers for methodological validation to prove that the extracted particles mainly come from your cells rather than the medium background. After validation passes, proceed to the formal experiment.

Whichever method you choose, the experimental design should include a blank control: the same volume of serum-containing medium without cells, extracted under the same culture conditions and ultracentrifugation workflow, and the particle count/protein amount measured as background. This allows assessment of the contribution of the serum background. It is recommended to use the same batch of serum and the same extraction method throughout, without changing them midway, otherwise the data will drift. If you do not use exosome-depleted FBS, reviewers may ask about this when you write your paper, and you will need to clearly describe the depletion method and validation data. Overall, performing ultracentrifugation yourself or buying domestic alternatives is a more realistic path when the budget is limited.

Serum-free starvation is a commonly used alternative, but it causes stress to cells, potentially inducing apoptosis or autophagy, altering exosome yield and cargo, and even contaminating the preparation with apoptotic bodies. It is recommended to first test different starvation durations, such as 12, 24, and 48 hours, and check cell viability before collection, keeping it above 85% if possible; before switching to serum-free medium, gently wash with PBS 2-3 times to reduce residual serum. Tolerance varies greatly among different tumor cells; some cells show a significant drop in viability after 24 hours of starvation and require optimization. Do not directly apply others' protocols.

If you are performing exosome extraction rigorously, regular FBS will indeed introduce bovine-derived exosomes, which mix with tumor cell exosomes and affect particle size, protein, and RNA analysis. If you are only doing WB to validate markers, the background may still be acceptable; but for proteomics, RNA sequencing, or functional experiments, it is recommended to switch to exosome-depleted FBS, or at least ultracentrifuge regular serum. You should also set up a cell-free medium control to see how many particles come from the reagent background. Otherwise, it will be very troublesome when you discover bovine contamination only after the data come out.