The Impact of Different Isolation Methods on Extracellular Vesicle Yield and Composition in human Breast Milk: A Pilot Study
- 1University of Auckland
Abstract
Background: Extracellular vesicles (EVs) play a key role in intercellular communication and are present in diverse physiological fluids, including breast milk (BM). Evidence has suggested that BM‐derived EVs (BMEVs) impact the health and well‐being of infants. To gain insights into the biochemical composition and functionality of BMEVs, effective isolation with minimal contamination is essential. Various methods have been utilised for BMEV isolation, each with inherent advantages and limitations. This pilot study compares three isolation methods—ultracentrifugation (UC), size exclusion chromatography (SEC), and multimodal chromatography with a charged core bead Capto Core column (CC)—to determine the most efficient approach for enriching BMEVs from small BM sample volumes.
Methods: BMEVs were isolated from samples collected from three donors using UC, SEC, and CC. BMEV yields and cargo were evaluated using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), BCA protein quantification, western blotting, proteomics, and small RNA sequencing.
Results: NTA showed similar particle concentrations and size distributions across all three isolation methods, and TEM validated the presence of EVs. Particle‐to‐protein ratios differed significantly across isolation methods, with SEC showing a significantly higher ratio than UC. Western blot and proteomics analyses showed greater enrichment of EV‐associated proteins in SEC and CC compared to UC, while CC showed the lowest abundance of BMEV contaminants (alpha‐S1‐casein, lactoferrin and α‐lactalbumin). UC captured a distinct miRNA profile including higher enrichment of hsa‐miR‐16‐5p compared to other methods.
Conclusion: Based on our findings, the CC isolation method may serve as a viable approach for isolating an EV‐enriched fraction from BM. CC was more effective at reducing co‐isolated contaminants compared to UC or SEC, while maintaining enrichment of EV‐associated proteins. Additionally, CC‐based isolation is cost‐effective, user friendly, and highly scalable, thus making it a potential practical and efficient solution for both routine research and large‐scale clinical applications.
Related
- Two-Dimensional Size-Exclusion Chromatography-Based Isolation and Characterization of Plasma-Derived Extracellular Vesicle Subpopulations from Prostate Cancer Patients
- Protocol for endothelial cell-derived extracellular vesicle isolation and characterization.
- Optimization of seminal extracellular vesicle isolation protocols for cancer biomarker analysis using proteomic profiling.
- Chemokine CXCL12 Mediates Transcription Factor HSF1-Dependent Small Extracellular Vesicle Secretion Through Different Endosomal Stages
- Isolation of blood-derived extracellular vesicles for disease diagnosis: Methods, challenges, and applications
BibTeX
@article{liu2026impact,
title = {{The Impact of Different Isolation Methods on Extracellular Vesicle Yield and Composition in human Breast Milk: A Pilot Study}},
author = {F. Liu and H. H. Phua and C. Blenkiron and A. Artuyants and D. Shea and M. J. Middleditch and X. Dong and G. Toldi and M. H. Vickers and B. B. Albert and F. Ramzan},
journal = {Journal of extracellular biology},
year = {2026},
month = nov,
doi = {10.1002/jex2.70200},
url = {https://europepmc.org/article/PMC/PMC13730444},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - The Impact of Different Isolation Methods on Extracellular Vesicle Yield and Composition in human Breast Milk: A Pilot Study
AU - F. Liu
AU - H. H. Phua
AU - C. Blenkiron
AU - A. Artuyants
AU - D. Shea
AU - M. J. Middleditch
AU - X. Dong
AU - G. Toldi
AU - M. H. Vickers
AU - B. B. Albert
AU - F. Ramzan
PY - 2026
DA - 2026/11/01
JO - Journal of extracellular biology
DO - 10.1002/jex2.70200
UR - https://europepmc.org/article/PMC/PMC13730444
AB - Background: Extracellular vesicles (EVs) play a key role in intercellular communication and are present in diverse physiological fluids, including breast milk (BM). Evidence has suggested that BM‐derived EVs (BMEVs) impact the health and well‐being of infants. To gain insights into the biochemical composition and functionality of BMEVs, effective isolation with minimal contamination is essential. Various methods have been utilised for BMEV isolation, each with inherent advantages and limitations. This pilot study compares three isolation methods—ultracentrifugation (UC), size exclusion chromatography (SEC), and multimodal chromatography with a charged core bead Capto Core column (CC)—to determine the most efficient approach for enriching BMEVs from small BM sample volumes. Methods: BMEVs were isolated from samples collected from three donors using UC, SEC, and CC. BMEV yields and cargo were evaluated using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), BCA protein quantification, western blotting, proteomics, and small RNA sequencing. Results: NTA showed similar particle concentrations and size distributions across all three isolation methods, and TEM validated the presence of EVs. Particle‐to‐protein ratios differed significantly across isolation methods, with SEC showing a significantly higher ratio than UC. Western blot and proteomics analyses showed greater enrichment of EV‐associated proteins in SEC and CC compared to UC, while CC showed the lowest abundance of BMEV contaminants (alpha‐S1‐casein, lactoferrin and α‐lactalbumin). UC captured a distinct miRNA profile including higher enrichment of hsa‐miR‐16‐5p compared to other methods. Conclusion: Based on our findings, the CC isolation method may serve as a viable approach for isolating an EV‐enriched fraction from BM. CC was more effective at reducing co‐isolated contaminants compared to UC or SEC, while maintaining enrichment of EV‐associated proteins. Additionally, CC‐based isolation is cost‐effective, user friendly, and highly scalable, thus making it a potential practical and efficient solution for both routine research and large‐scale clinical applications.
SN - 2768-2811
ER -