Insights into the variability of extracellular vesicle preprocessing and extraction methods

Extracellular vesicles (EVs) are minuscule lipid vesicles that serve as vital messengers, shuttling cargo between cells and playing pivotal roles in various physiological processes. Understanding EVs and their cargo could offer valuable insights into both health and disease conditions. However, before their potential can be fully realized, EVs must undergo preservation, extraction, and often preconcentration processes. In a recent study, researchers at McGill University conducted a comprehensive analysis comparing different methods for EV preservation, extraction, and characterization, shedding light on the strengths and weaknesses of each approach.

Comparing Preservation Agents

The study began by comparing plasma preservation agents, focusing on ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants. Results showed that the choice of preservation agent varied in its effectiveness depending on the extraction method employed, highlighting the importance of considering preanalytical parameters in EV research.

Assessing EV Extraction Methods

The researchers evaluated four different EV extraction methods: ultracentrifugation (UC), size-exclusion chromatography (SEC), centrifugal filtration (LoDF), and accoustic-sorting (AcS). Each method was benchmarked based on various parameters, including integrity, size distribution, concentration, purity, and expression profiles of EV-associated proteins, as well as throughput, time-to-result, and cost.

Key Findings and Implications:

  • Ultracentrifugation produced a high yield of EVs with low contamination, making it a reliable method for EV extraction.
  • Size-exclusion chromatography (SEC) emerged as a low-cost, fast, and easy-to-implement method, although it exhibited lower purity of EVs.
  • Centrifugal filtration (LoDF) and accoustic-sorting (AcS) showed compatibility with process automation, small volume requirement, and rapid processing times.
  • However, LoDF was prone to clogging and sample contamination when using plasma, while AcS yielded high-purity EVs but with a lower extraction yield.
  • Analysis of protein profiles revealed that different extraction methods extracted distinct subpopulations of EVs, underscoring the importance of method selection in EV research.

Overall, this study provides valuable insights into the strengths and weaknesses of various EV extraction methods, as well as the variability in concentration, purity, and EV expression profiles obtained. By considering preanalytical parameters and selecting appropriate extraction methods, researchers can better address the diversity of EVs and harness their potential as clinically actionable indicators. This research marks a significant step forward in advancing our understanding of EV biology and its implications for disease diagnosis and treatment.

Alexandre L, Shen ML, de Araujo LOF, Renault J, DeCorwin-Martin P, Martel R, Ng A, Juncker D. (2024) Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles. ACS Sens [Epub ahead of print]. [abstract]

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