Enrichment of plasma-derived extracellular vesicles from astrocytes
Extracellular vesicles (EVs) are nanoparticles that are secreted by all cells in the body. EVs carries multiple cellular components, such as genetic material, proteins and lipid metabolites that often reflect the content of their cell of origin. They are involved in a wide range of functions by acting on and transferring their contents to other cells under physiological and pathological conditions. Given their small size, EVs are able to diffuse into all biological fluids such as plasma and cerebrospinal fluid (CSF), from where they can be selected based on the specific surface markers they express, accurately reflecting the specific tissue environment from which they originate. Thus, EVs represent an excellent non-invasive diagnostic tool, and can be considered as a liquid biopsy for biomarker discovery. This aspect is of particular interest in neurology, given the difficulties in studying brain analytes in accessible fluids other than CSF. In this regard, astrocytes have rising interest, since they are at the intersection of neuro-vascular communication, performing essential functions in the brain. In neurodegenerative diseases, reactive astrocytosis can lead to altered vesicular cargo, facilitating disease progression, and analysis of the EV cargo they release may lead to the discovery of biomarkers.
What has been done in this study
A limitation of the use of these vesicles in research is that there is a lack of standardised methodological protocols for their isolation. In this article, we present a detailed protocol for the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. This protocol consists of a first isolation of total EVs by polymer precipitation, followed by a second step of immunoprecipitation of ADEVs.
We were able to optimize a detailed and simple method for the enrichment of ADEVs from human plasma. The protocol was validated, confirming the presence of vesicular and astrocyte markers in the ADEV fraction, as well as the presence of contaminating lipoproteins and verifying their size and morphology by nanoparticle tracking analysis and electron cryo-microscopy. The applicability of this protocol was also verified by quantifying inflammatory markers in these ADEVs, in which all the cytokines measured were detected.
Relevance of the study
Given the important functions of astrocytes, the study of the content of ADEVs could be an interesting platform for biomarker discovery and exploration of inflammatory pathways in the brain in a non-invasive manner.