Exosomes are small microvesicles, derived from the late endosome, most often described in the literature to be less than 120 nm, released by all cell types, and proven to be involved in cancer metastasis1-3. Exosome characterization and analysis comprise a fast, evolving research area even though their biological function has yet to be completely elucidated. Exosomes contain proteins, lipids, and microRNA capable of regulating an assortment of target genes. Recent studies have suggested that exosomes can serve as biomarkers for future clinical and diagnostic use in not only cancer but many other human diseases4. Exciting new findings have implicated exosomes in cardiovascular diseases5-7, autoimmune syndromes8, and neurodegenerative disorders such as Alzheimer’s9 and Parkinson’s10 disease, in addition to infectious diseases such as tuberculosis11, diphtheria12, and even HIV13.
Much of the research involving exosomes uses a cell culture platform, although exosomes are routinely isolated from other bodily fluids such as plasma, serum, urine, and breast milk. In cell culture, fetal bovine serum (FBS) is usually incorporated into media, despite FBS containing extremely high levels of innate bovine extracellular vesicles which complicate downstream analyses. There has recently been a call for the importance of depleting cell culture media of contaminating bovine exosomes14 within the research community. In order to deplete FBS of bovine exosomes, researchers routinely turn to ultracentrifugation, due to the simplicity and efficiency of the process. In this process, large volumes of FBS can easily be centrifuged at high speeds to eliminate native extracellular vesicles typically in an overnight spin. However, there are also commercially available products which have pre-conditioned the media to be depleted of exosomes and other microvesicles. The process here is proprietary and is dependent on the manufacturer but is often very costly compared to source FBS. Here, we will explain the process for “home-brewing” exosome-depleted FBS by ultracentrifugation and compare the cell viability and media depletion percentage from two separate cell lines after treatment with different media types. Suggested ways to isolate exosomes of interest from cell culture with an example centrifugation protocol will also be discussed.