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Table 2 Methods of exosome isolation

From: The versatile role of exosomes in human retroviral infections: from immunopathogenesis to clinical application

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