Introductory lecture on EV Isolation from Chris Gardiner presented at ISEV2014. For more details go to www.isev.org

1. MEDICAL SCIENCES DIVISION: UNIVERSITY OF OXFORD
Nuffield Department of Obstetrics and Gynaecology
Isolation of extracellular vesicles
Chris Gardiner: University of Oxford

2. Pre-analytical variables
represent the largest single
source of variability in the
analysis of extracellular vesicles

3. Current techniques for EV isolation
• Ultracentrifugation
• Density gradient centrifugation
• Immuno-magnetic bead capture
• Ultrafiltration
• Precipitation
• Microfluidics
• ELISA
• Field flow fractionation
• Affinity purification
• Size exclusion chromatography
• HPLC…………………………..

4. Considerations in selecting isolation method
• Type of sample
• What are you going to do with the EV?
• How important is yield?
• How important is purity?
• How important is processing time?
• What is your starting sample volume?

5. Sample type
• Starting vesicle concentration
– Blood > Plasma > Conditioned media
• Starting volume
• Sample viscosity
• Sample complexity
– Plasma proteins
– Tamm–Horsfall protein
– “exosome-free serum” in media
– lipoproteins

6. What are you going to do with the EV?
• Flow cytometry, Nanoparticle Tracking Analysis,
Tuneable Resistive Pulse Sensing, etc.
• Western blotting
• Proteomic analysis
• RNA analysis
• Functional analysis
• Therapeutic applications

7. Yield versus purity
• There is always a trade-off!
• Do you want to isolate all EV in the sample, just
exosomes, or a specific population of EV?
• Is your protein/RNA of interest actually in your
vesicles?
• Is this important to you?

8. Standardised protocol for preparing platelet poor plasma
2500 ×g for 15 min at RT within 1 hour of collection prior to snap
freezing in liquid nitrogen and storage at -80ºC
Efficient removal of residual platelets reduces inter-laboratory variation
by preventing ex-vivo formation of EV during freeze/thawing.
Excellent for intended use, i.e., multicentre flow-cytometric studies of
plasma EV
But double centrifugation reduces plasma EV by ~80% compared with
whole blood (by FCM)
Not suitable for isolating rare populations (e.g. Tumour vesicles) or
larger microvesicles

9. Pros and cons
Method Pros Cons
Ultracentrifugation Relatively straightforward Purity/EV integrity
Density centrifugation High purity Slow, sucrose toxicity
Ultrafiltration Quick Low purity
Immuno-magnetic bead Specific, high purity Prior knowledge of EV
characteristics required
Affinity purification Relatively specific, high
purity
Prior knowledge of EV
characteristics required
Chromatography High purity Specialised equipment
Microfluidics Specific, high purity Prior knowledge of EV
characteristics required
Field flow fractionation Quick ? purity
Precipitation techniques High yield Low purity

10. Conclusions
• Sample type, volume, viscosity and complexity must
be considered
• Decide what you are trying to achieve and what is
most important to you
• Do not change technique during a study
• There is no “one size fits all” method
• We may need several standardised isolation methods
to achieve our aims

11. Future directions
• ISEV are discussing a multicentre comparative
evaluation of commercially available EV isolation kits
• Problems
– Funding??
– Would the manufacturers provide the kits free of
charge?
– Which samples should be tested?
– Which methods should we use to characterise the
purified EV?
• A survey will be sent out in the near future