Table 4 Comparison of EVs isolation approaches

Centrifugation-based methods

 Ultracentrifugation

Centrifugal force

(1) Cost-effective;

(2) No supplement required

(1) Low EV yield;

(2) Disruption and aggregation of EVs;

(3) Coisolation;

(4) Time- and equipment-consuming;

(5) Low reproducibility

 Multiple-step centrifugation

EV isolation by sedimentation

(1) Well validated;

(2) Suitable for a large volumeof sample;

(3) No additional reagents required

(1) Efficiency varies among different biological Sources;

(2) EV integrity may be compromised;

(3) Time consuming;

(4)Requires an expensive ultracentrifuge for small EV

 Density gradient ultracentrifugation

EV isolation by size and density

(1) Efficient at preserving EV characteristics;

(2) Suitable for downstream analysis;

(3) High purity

(1)Time consuming;

(2)Subjected to operator-based variability;

(3)Low yield;

(4)Requires expensive ultracentrifuge

Precipitation-based methods

 Precipitation with cationic polymers

Sedimentation of EVs using polymers and sedimentation

(1) Simple;

(2) Not equipment- consuming;

(3) High EV recovery

(1) Low purity;

(2) Contamination with polymers and non-EV particles;

(3) Low reproducibility

Size-based methods

 Ultrafiltration

Filtration through semi-permeable membrane

(1) Medium-to-high yield;

(2) Simple;

(3) scalable;

(4) fast

(1) Possible contamination with proteins;

(2) Non-specific binding of EVs to membrane;

(3) Possible EV damage

 Tangential flow filtration

Filtration through a semi-permeable membrane with tangential flow

(1) High yield;

(2) Scalable;

(3) High purity;

(4) High reproducibility;

(5) Optimal as pre-concentration method

(1) Special equipment required;

(2) Contamination with large proteins and non-EV particles

 Asymmetrical flow-field-flow fractionation

Separation in parabolic flow according to diffusion capacity

(1) High reproducibility;

(2) Separation of heterogeneous fractions

(1) Special equipment required;

(2) Limited scalability

Chromatography methods

 Anion-exchange chromatography

EV adsorption onto positively-charged sorbents

(1) High yield;

(2) High purity;

(3) High reproducibility

(1) Requires pre-concentration for large volumes;

(2) Contamination with non-EV particles

(3) Requires an additional buffer- exchange step

 Hydrophobic chromatography

Adsorption of uncharged vesicles onto hydrophobic sorbent in high-salt buffer

(1) High purity;

(2) Fast;

(3) Low cost;

(4) Scalable

(1) Variable EV yield

Affinity-based isolation methods

 Beads conjugated with antibodies against tetra- spanins

EV isolation via highly specific interactions with surface markers

(1) Highest purity;

(2) High recovery;

(3) Fast;

(4) Not equipment-intensive; selective for EVs

(1) High cost;

(2) Highly limited scalability for most approaches