Isolating Small Extracellular Vesicles from Small Volumes of Blood Plasma using size exclusion chromatography and density gradient ultracentrifugation: A Comparative Study

Reviewer #1 (Public Review):

Summary:
In their manuscript, Kong Fang et al describe a robust pipeline for the isolation of small extracellular vesicles through a combination of size exclusion chromatography and miniaturized density gradient separation. Subsequently, they prove that the method is reproducible and suitable for small-volume operations while at the same time not compromising the quality of vesicles.

Strengths:
The paper narrates a robust method for purifying high-quality sEVs from small amounts of blood plasma. They also demonstrate that through this approach, they can derive sEVs without compromising the protein composition, integrity of the vesicles, or contamination with other proteins or lipids.

Weaknesses:
The paper is a nice summary of how to enrich sEVs from blood samples. Although well performed and substantiated with data, the paper primarily deals with method development and optimisation.

Reviewer #2 (Public Review):

Summary:
In this work, the authors manage to optimize a simple and rapid protocol using SEC followed by DGCU to isolate sEVs with adequate purity and yield from small volumes of plasma. Isolated fractions containing sEVs using SEC, DGCU, SEC-DGCU, and DGCU-SEC are compared in terms of their yield, purity surface protein profile, and RNA content. Although the combined use of these methodologies has already been evaluated in previous works, the authors manage to adapt them for the use of small volumes of plasma, which allows working in 1.5 mL tubes and reducing the centrifugation time to 2 hours.

The authors finally find that although both the SEC-DGCU and DGCU-SEC combinations achieve isolates with high purity, the SEC-DGCU combination results in higher yields.

This work provides an interesting tool for the rapid obtention of sEVs with sufficient yield and purity for detailed characterization which could be very useful in research and clinical therapy.

Strengths:
-The work is well-written and organized.
-The authors clearly state the problem they want to address, that is, optimizing a method that allows sEV to be isolated from small volumes of plasma.
-Although these methodologies have been tested in previous works, the authors manage to isolate sEVs of high purity and good performance through a simple and fast methodology.
-The characteristics of all isolated fractions are exhaustively analyzed through various state-of-the-art methodologies.
-They present a good interpretation of the results obtained through the methodologies used.

Weaknesses:
-Lack of references that support some of the results obtained.
-Although this work focuses on comparing different techniques and their combinations to find an optimal option, the authors do not use any statistical method that reliably shows the differences between these techniques, except when repeatability is measured.