Current methods for the isolation of extracellular vesicles

Abstract

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

Figure 1. A typical ultracentrifugation protocol.

In consecutive rounds of centrifugation and pouring off, the RCF (g) and the centrifugation time are increased to pellet smaller particles. After each of the first three centrifugations, pellets that contain dead cells and cell debris are discarded, and the supernatant is kept for the next step. In contrast, after the 100 000 g centrifugations, pellets (containing EVs) are kept, and supernatants are discarded. The pellets are resuspended in phosphate buffered saline (PBS) for further analysis.

Figure 2

Transmission electron microscopy (TEM) characterization of human serum derived EVs isolated using (A) conventional differential centrifugation and (B) sucrose gradient centrifugation.(A) EVs isolated from human serum expressing CD63 transmembrane protein, which is believed to be exosome/microvesicles marker. There is more immuno-gold labeled protein aggregate in the background in this sample prepared via conventional differential centrifugation protocol. (B) EVs isolated from human serum expressing CD63 transmembrane, protein which is believed to be exosome/microvesicles marker. The background is neat, with minimal amount of protein aggregates after purification with sucrose gradient centrifugation.

Figure 3. Antibody-coated magnetic beads.

This illustration demonstrates how antigens of extracellular vesicles (EVs) bind to the antibodies of coated magnetic beads, which also bind the magnetic sensors, called Hall sensors, on the surface of the chip.