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Updated: Sep 23, 2025
Extraction of Extracellular Vesicles from Whole Tissue
Published on: February 7, 2019
Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell-Cell
Venkatesh Kumar Chetty1, Jamal Ghanam1, Srishti Anchan1
1Department of Pediatrics III, University Hospital Essen, 45147 Essen, Germany.
Abstract:
Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell-cell communication in cancer.
Insights
Researchers developed a new method to isolate small extracellular vesicles (sEVs) and their associated DNA. This technique reveals how EV-DNA influences cell communication, particularly in cancer.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Small extracellular vesicles (sEVs) are crucial for intercellular communication.
- Studying DNA within sEVs is challenging due to difficulties in separating them from free DNA and apoptotic bodies.
Purpose of the Study:
- To develop a robust method for isolating sEVs and their associated DNA.
- To investigate the distribution and cellular uptake of DNA carried by sEVs.
- To explore the role of EV-DNA in cell-cell communication, especially in cancer.
Main Methods:
- Combined tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU) for sEV isolation.
- Utilized high-resolution 3D imaging and super-resolution microscopy.
- Analyzed EV-DNA interaction with cellular components and recipient cells.
Main Results:
- The TSU method efficiently separated sEVs and enriched dsDNA from host genome and mitochondria within vesicles.
- EV-DNA was shown to cross recipient cell membranes, entering cytoplasm and nucleus.
- EV-DNA was observed to interact with cGAS/STING inflammatory sensors and Rab5/Rab7 endosomal proteins.
- Leukemia-derived EV-DNA was found to communicate with mesenchymal stromal cells.
Conclusions:
- The TSU method provides a reliable tool for studying EV-DNA.
- EV-DNA plays a significant role in intercellular communication, impacting cellular pathways.
- Understanding EV-DNA's role is critical for cancer research and therapeutic strategies.
