Separating by size and density
Differential ultracentrifugation spins a sample at increasing speeds so that cells, debris and finally vesicles settle in turn. It is widely used and needs little special chemistry, but it is slow, hard to scale and can damage vesicles or leave contaminants behind.
Density gradients refine the result by separating vesicles from other particles of a similar size.
Filtering and column methods
Tangential flow filtration passes fluid across a membrane so that small molecules pass through and vesicles are retained. It scales well and is gentle, which makes it attractive for larger volumes.
Size-exclusion chromatography lets larger particles move through a column faster than small ones. It gives clean vesicles with good integrity, though it dilutes the sample and handles modest volumes.
Capturing by identity
Affinity methods use antibodies or other binders to pull out vesicles carrying a chosen surface marker. They are highly selective, which is useful for sub-populations, but cost and yield can limit them.
Precipitation kits are simple and fast, yet they tend to bring along other material and so suit screening better than precision work.
Why combining methods wins
Because each method has a weak point, many programmes use orthogonal steps, for example filtration followed by chromatography, and then confirm purity analytically.
Whatever the route, the preparation should be described by its method and its measurements so another laboratory can reproduce it.
- Ultracentrifugation, filtration, chromatography and affinity capture each have strengths and limits.
- Combining different methods usually gives a purer and more reliable result.
- Always describe a preparation by both method and measurements.
Common questions
Which isolation method gives the purest exosomes?
No single method is best. Combining different principles, such as filtration followed by chromatography, usually gives a purer result than any one step.
Why is ultracentrifugation so common?
It needs little special chemistry and is available in many laboratories, although it is slow and hard to scale.
How do you know an isolation worked?
By measuring the preparation, for example particle size and concentration, marker proteins and purity, and recording the method alongside the data.
