Table 2.

Methods of exosome isolation

Isolation methods	Mechanism	Advantages	Disadvantages
Ultracentifugation	The method consists of a series of centrifugation to remove cells and debris and precipitate exosomes based on size and density	This method is the standard and gives pure exosomes and viruses from bio-fluids and cell culture media	The efficiency of the method is low for the isolation of exosomes from plasma and serum. Overlapping densities between viruses and exosomes
Density gradient centrifugation	This method is ultracentrifugation aimed at an iodixanol or sucrose based on a density gradient	Through this method small and low-density exosomes are isolated from particles, other vesicles, and contaminants	Sensitive to the centrifugation time and needs more minuteness. Overlapping densities between viruses and exosomes
Filtration	This method uses ultrafiltration membranes to isolate exosomes from proteins aggregation and other macromolecules	In this method, small particles and soluble molecules separate from exosomes. Exosomes are concentrated on the filtration membrane	Exosomes may be lost due to adhering to the filtration membranes. Besides, the additional force may be deformed or damaged exosomes. Only useful when starting with large volumes Overlapping densities and size between viruses and exosomes
Size exclusion chromatography	In this method, using a column packed with porous polymeric beads and size-exclusion chromatography macromolecules are separated based on their size. It applies	In this method, large and small molecules are separated. This method can separate exosomes from viruses. Also, the structure of isolated exosomes is not changed by the shearing force	The method is time-consuming. Overlapping size between viruses and exosomes
Immunological separation	Several immunological methods such as Magnetic beads bound to the specific antibodies and the ELISA-based separation method	This method selectively isolates exosomes or subpopulation of exosomes. This method can separate exosomes from viruses Also, it is applicable for the characterization and quantitation of exosomal markers	Small sample volumes are required and the isolated vesicles may fail the functional activity as well as can require initial concentration step
Polymer-based precipitation	This method which is prepared as commercial kits comprises mixing the biofluids with precipitation solution, incubation step, and low-speed centrifugation	This method has a mild effect on isolated exosomes and the usage of neutral pH	In this method, exosomes are isolated with contamination and the presence of the polymer material may affect downstream analysis. Overlapping size between viruses and exosomes
Isolation by sieving	In this method, exosomes are isolated by sieving through a membrane and filtration by electrophoresis or pressure	Fairly short isolation time and isolated exosomes are pure	Low recovery of isolated exosomes. Overlapping size between viruses and exosomes
Microfluidics-based techniques	This method is microscale isolation based on exosomes immunoaffinity, size, and density	This method is a low cost, fast, portable, and high portability	Limitation in standardization and large scale tests on clinical samples, Limitation in method validation, moderate to low sample capacity. Overlapping size between viruses and exosomes
nanoFACS	Similar to fluorescence assisted cell sorting (FACS), nanoscale flow cytometry and nanoFACS are meant to identify and sort EVs subpopulations based on a heterogeneous input population	This method can separate exosomes from viruses based on indirect fluorescence labeling or de novo labeled proteins (like GFP-HSV-1 fusion proteins, and GFP-Gag). It can be employed to rapidly characterize heterogeneous input mixtures without the need to concentrate them first	Only certain flow cytometers calibrated for nanoFACS Sample typically requests to be diluted earlier nanoFACS and may be additional diluted post sorting