Extracellular Vesicle Preparation

Extracellular vesicles (EVs), such as exosomes, are fascinating particles that can deliver proteins, lipids, and RNAs to distant target cells. Critical to understanding EV function is reliable enrichment of pure fractions. Many investigators use ultracentrifugation, which is lengthy and labor intensive. However, size exclusion, using low-MWCO ultrafiltration membranes, is becoming more and more widely used and cited in literature.

In this learning center, you'll find resources to help you make sure that your EV preparations are reproducible and accurate. You can also learn how prepare extracellular vesicles with:

  • Rapid, centrifugal ultrafilter-based method using Amicon® Ultra filters
  • Equivalent yield/quality to ultracentrifuge preparations
  • Better yield/quality than polymeric precipitation-based techniques

Read Protocol

Order Amicon® Ultra centrifugal filters

Use the Amicon® Ultra selector tool to determine appropriate molecular weight cutoff (MWCO) and device size.

Supplementary Protocols: Monitoring Enrichment, Detecting Protein Cargo and Relative Quantitation

Also in this learning center, you will learn new techniques for streamlined analysis of your EV preparations. The Supplementary Protocols section shows you how to:

Background Information: Diverse Methods Used for Exosome and Extracellular Vesicle Preparation

Given the many methods available for EV preparation, it is useful to consider the methods in light of the scientific question you are asking and downstream analytical techniques. This table shows a snapshot of EV preparation methods and some of their advantages and disadvantages. We hope it will help you better plan your extracellular vesicle and exosome research!

Table 1. Extracellular vesicle preparation methods, listing some advantages and disadvantages.

EV Preparation Method Pros Cons

Ultracentrifugation (sometimes with sucrose gradient)
  • Reproducible
  • Relatively accurate
  • Generates functional vesicles
  • Time-consuming
  • Requires use of large, specialized shared equipment (ultracentrifuge)
  • Prep may also include other soluble proteins and aggregates
  • EVs may get damaged

Affinity Purification
  • High selectivity
  • Relatively fast and simple
  • Lower yields
  • Markers may not be representative of the targeted EV population
  • Solution conditions may be incompatible with downstream assays

Ultrafiltration (i.e. size exclusion through low MWCO membranes)
  • Generates functional vesicles, as reported for preparation of therapeutic EVs
  • Relatively fast and simple
  • Depending on the membrane and force applied, EVs may be deformed or broken up

Polymeric Precipitation
  • Relatively fast and simple
  • Contamination by other soluble proteins