Cells not only actively secrete membrane vesicles but also passively release protein, RNA, or membrane complexes/aggregates, especially when cells undergo necrotic or apoptotic cell death (1). In cell culture supernatant, complexes/aggregates released from dying cells are present together with exosomes secreted from other healthy cells. These aggregates remain in cell culture supernatants even after centrifugation at 10,000 g according to standard extracellular vesicle preparation protocols, and aggregates and exosomes co-sediment during 100,000 g centrifugation. Hence, aggregates contaminate these crude exosome pellets. The contamination of protein/RNA/membrane aggregates is especially problematic when cell death is induced during in vitro cell culture. For example, apoptosis and activation are both induced when macrophages are stimulated by LPS or when T cells are stimulated by α-CD3 and α-CD28 antibodies in vitro. Therefore, an optimized strategy for separation of aggregates from exosomes is critical for studying exosomes secreted from immune cells under stimulated conditions.
Exosomes have been found to float at densities ranging from 1.15 to 1.19 g/ml on sucrose gradients. By comparison, vesicles purified from the endoplasmic reticulum float at 1.18 to 1.25 g/ml, vesicles from the Golgi at 1.05 to 1.12 g/ml (2). The density of protein aggregates is 1.22 g/ml (3). The differing density of the potential contaminants of exosomes make sucrose gradient centrifugation an effective strategy for further purification of exosomes from sedimented crude exosome pellets.