Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation

TLDR

Exosomes are 50–100 nm extracellular vesicles released from endosomal compartments, while other membrane vesicles can be shed by plasma‑membrane budding and are often difficult to separate from exosomes. The authors sought to demonstrate that classical ultracentrifugation of mouse tumor‑cell conditioned medium yields at least two distinct vesicle populations by modulating exosome secretion with Rab27a inhibition. They inhibited Rab27a to alter exosome release and then purified vesicles by standard ultracentrifugation from the conditioned medium. Rab27a inhibition reduced secretion of conventional exosome markers (CD63, Tsg101, Alix, Hsc70) but not CD9 or Mfge8, and density‑gradient and EM analyses revealed CD9 and Mfge8 on vesicles ranging from 30 nm to >150 nm, indicating that common exosome preparations co‑purify heterogeneous vesicles from endosomal and plasma‑membrane origins.

Abstract

Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome‐derived vesicles. We propose that the most commonly used protocols for exosome preparations co‐purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles.