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Separation of small extracellular vesicles (sEV) from human blood by Superose 6 size exclusion chromatography. | LitMetric

AI Article Synopsis

  • Extracellular vesicles (EVs) show promise as biomarkers for liquid biopsy, but current methods struggle to isolate them effectively from complex biofluids like plasma and serum.
  • Traditional isolation techniques often lead to contamination with lipoproteins, making it difficult to accurately assess the number and content of EVs.
  • The introduction of Fast Protein Liquid Chromatography (FPLC) with Superose 6 offers a better solution to isolate small EVs while minimizing contamination, highlighting their potential for detecting disease-associated biomarkers, especially in pancreatic cancer patients.

Article Abstract

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV-based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell-free plasma or serum. Because available one-step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2-step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV- and tumour-biomarkers in pure FPLC-derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases-associated biomarkers in pure EVs, supporting the feasibility of using single-vesicle analysis for liquid biopsy.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11497763PMC
http://dx.doi.org/10.1002/jev2.70008DOI Listing

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