Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

Reine Khoury; Laura Montermini; Nadim Tawil; Corina Nagy

doi:10.3791/70894

Method Article

Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

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DOI:

10.3791/70894

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June 23rd, 2026

Reine Khoury¹^,² , Laura Montermini³ , Nadim Tawil³ , Corina Nagy¹^,²^,⁴

¹Integrated Program in Neuroscience, McGill University, ²McGill Group for Suicide Studies, Douglas Mental Health University Institute, ³Center for Applied Nanomedicine, Research Institute of the McGill University Health Center, ⁴Department of Psychiatry, McGill University

Corresponding Authors: Corina Nagy <corina.nagy@mcgill.ca>

Summary

This protocol describes the isolation of small extracellular vesicles from murine skeletal muscle and bone marrow using enzymatic digestion and size-exclusion chromatography, enabling reproducible recovery of vesicle-enriched fractions from structurally complex, low-yield tissues for downstream molecular analysis.

Abstract

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, lipids, and ribonucleic acids (RNAs). Isolation of sEVs from solid tissues such as skeletal muscle (SkM) and bone marrow (BM) remains challenging due to low yield and contamination from non-vesicular components. This protocol describes a reproducible workflow for isolating sEVs from mouse SkM and BM. SkM is enzymatically digested, whereas BM is processed directly and subsequently subjected to differential centrifugation and size-exclusion chromatography (SEC). For SkM, the void volume (~2.5 mL) is discarded, and the subsequent ~1.6 mL is collected as EV-enriched fractions, typically subdivided into 400 µL sequential fractions (F1–F4). For BM, a 700 µL void volume is discarded, followed by collection of an ~850 µL EV-enriched fraction, which can be subdivided into ~170 µL sequential fractions (F1–F5) and pooled based on EV marker enrichment. Using this approach, sEVs can be isolated from small tissue volumes (approximately 500 µL from a single quadriceps and 150 µL from pooled femur and tibia BM), yielding ~10⁸ particles·mL⁻¹·mg⁻¹ tissue with particle diameters predominantly <200 nm. Vesicle integrity and purity are validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting for canonical EV markers, with additional characterization by single-particle interferometric detection to quantify tetraspanin-defined vesicle subpopulations. This method enables reproducible isolation of high-purity sEVs from structurally complex tissues using minimal input material, supporting downstream molecular and functional analyses of tissue-derived vesicles.

Introduction

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, lipids, and nucleic acids¹. They are released by all cell types and are present in virtually all biological fluids, where they function as key carriers of bioactive cargo, including peptides and micro ribonucleic acids (RNAs)². Their cargo is selectively sorted, enabling precise delivery of specific signals³, and their vesicular structure allows them to bypass the typical gradient and dilution constraints of freely secreted factors by delivering concen....