Extracellular vesicles (EVs) are nanometer-size particles (30 nm to 1000 nm) that can be released by cells and are no longer capable of reproduction. These particles have a lipid bilayer membrane and serve as transporters for various biomolecules. These biomolecules assist in communication, which is why they play an increasingly important role in medical research. In the future, they will be used as biomarkers for a variety of diseases as well as in therapy of diseases by packing substances and transporting them to specific target cells.
The ABPCI core facility has specialized in protocols for isolating and characterizing extracellular vesicles from cell culture supernatants.
Isolation of EVs
EVs can be detected in nearly all body fluids. The ABPCI core facility has specialized in the isolation of EVs from blood plasma and also cell culture supernatants. Depending on the area of application, a choice can be made between ultracentrifugation (UC) and size-exclusion chromatography (SEC).
In 2018 the International Society for Extracellular Vesicles issued a recommendation on how EVs should be isolated and characterized in order to guarantee reproducible and reliable data. When establishing protocols and planning testing, the ABPCI core facility always makes sure to incorporate these recommendations into their work. Link to recommendation
Size-exclusion chromatography (SEC)
SEC is becoming more and more popular in the community since this method promises good yields and very native EVs. In addition, isolation proceeds relatively quickly and can also be automated in further steps.
Currently EVs are isolated from platelet-free plasma (PFP) with Sepharose SEC columns that can be precipitated beforehand with polyethylene glycol (PEG).
Flow assisted cell sorting (FACS)
In addition to electron microscopy, FACS is presumably the most important method for investigating EVs. The great advantage here is that individual EVs can be investigated one after another for several markers at once. Due to the small size of the particles, special methods and settings should be used that are very different from "normal" FACS measurements.
Nano Tracking Analysis (NTA)
NTA is used to estimate the size distribution (30nm to 1000nm) and particle concentration of EVs in samples. The particles are excited by a laser. The Brownian motion of their particles is then filmed by a microscope. This footage is evaluated by a software program and the size as well as the concentration of the measured particles is calculated.
Western Blot (WB)
WB is mainly used to characterize the isolated EVs. The purity criterion is tetraspanin markers (CD9, CD63 and CD81), which must be detected. Depending on what biomaterial is used, there are negative markers that ideally cannot or can hardly be detected in samples. In the case of blood, it is the marker (e.g., apolipoprotein A1) for lipoprotein particles that often contaminates EV samples.