Does extracellular vesicle extraction require the use of extracellular vesicle serum? How to choose grading?

Exosome-depleted serum does not fall under traditional FBS grading standards; rather, it is a functionally processed type and should not be conflated with "low-endotoxin grade." When working with MSC supernatant exosomes, if background bands are excessive, ultracentrifugation of ordinary serum alone is often insufficient: bovine-derived exosomes, lipoproteins, and protein aggregates can all co-precipitate around 100,000g, and a single ultracentrifugation run may not clean them out completely—it may also cause co-precipitation loss of the target exosomes. It is recommended to prioritize commercial exosome-depleted FBS, and at minimum confirm that its process involves ultracentrifugation/precipitation removal and has been negatively validated for exosome markers such as CD63, CD81, and Calnexin. In-house processing is not unfeasible, but strict validation must be established, including NTA particle size and WB markers, and there is still a high probability of residual contamination.

From a practical standpoint, whether ordinary serum can "achieve equivalent results" through self ultracentrifugation—it is advisable not to set expectations too high. Exosome depletion requires multiple rounds of ultracentrifugation plus rigorous quality control; in the lab, variables such as rotor, centrifuge tubes, and temperature are numerous, and reproducibility is inferior to commercial batches. A more reliable strategy: when purchasing exosome-depleted serum, check whether the supplier provides a test report, such as NTA particle count below a certain background value and negative WB for exosome markers; after arrival, draw a bottle yourself and ultracentrifuge it, then measure the particle count to verify. If the budget is tight, exosome-depleted serum can be used only for the final two passages before collection, with ordinary serum used during the expansion phase to reduce consumption. Low endotoxin grade helps with cell status, but it is not the key to solving exosome background.

Carbon adsorption and dialysis types follow another logic. Carbon adsorption primarily removes small molecules such as lipophilic hormones, cytokines, and steroids, making it suitable for hormone-dependent cell or nuclear receptor research; dialysis types remove amino acids, nucleotides, and small-molecule metabolites, making them suitable for nutrient starvation, metabolic labeling, or drug treatment experiments. Neither is intended for exosome removal and cannot be used as a substitute. If your MSC exosome samples are precious, it is recommended to expand the cells in medium containing 10% exosome-depleted FBS, switch to serum-free or low-serum conditioned medium for 24-48 hours before collection, and then collect the supernatant. This can reduce serum-derived exosomes and protein complexes. For serum-free conditions, preliminary tests on cell viability and exosome yield should be performed in advance; do not switch entirely all at once.