Cell-specific isolation and functional analysis of primary cardiomyocyte-derived large extracellular vesicles from infarcted mouse hearts
- 1Université Paris Cité, PARCC, INSERM, F-75015 Paris, France
Abstract
Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.
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BibTeX
@misc{borowski2026cellspecific,
title = {{Cell-specific isolation and functional analysis of primary cardiomyocyte-derived large extracellular vesicles from infarcted mouse hearts}},
author = {L. Borowski and A. Ramazanova and P. Alayrac and V. Duval and M. Aubert and L. Pineau and L. Faranton and C. Knosp and E. Robidel and F. Dingli and D. Loew and J. Vilar and C. Boulanger and X. Loyer and J.-S. Silvestre and S. M. Camus},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.01.755891},
url = {https://www.biorxiv.org/content/10.64898/2026.10.01.755891v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Cell-specific isolation and functional analysis of primary cardiomyocyte-derived large extracellular vesicles from infarcted mouse hearts
AU - L. Borowski
AU - A. Ramazanova
AU - P. Alayrac
AU - V. Duval
AU - M. Aubert
AU - L. Pineau
AU - L. Faranton
AU - C. Knosp
AU - E. Robidel
AU - F. Dingli
AU - D. Loew
AU - J. Vilar
AU - C. Boulanger
AU - X. Loyer
AU - J.-S. Silvestre
AU - S. M. Camus
PY - 2026
DA - 2026/10/01
PB - bioRxiv
DO - 10.64898/2026.10.01.755891
UR - https://www.biorxiv.org/content/10.64898/2026.10.01.755891v1
AB - Extracellular vesicles (EVs) are membrane-bound particles secreted by all cells and serve as critical mediators of intercellular communication in both physiological and pathological contexts. However, elucidating the precise functions of endogenous EVs remains challenged by a fundamental technical bottleneck: current isolation methodologies depend on shared physical properties or generic surface markers, preventing the selective enrichment of cell-type-specific EVs within complex tissues. Consequently, EVs recovered from biofluids or tissue extracts represent heterogeneous populations, confounding the direct attribution of biochemical signatures and biological activities to distinct cellular origins. Here, we describe a versatile genetic-labeling and affinity-based purification strategy that enables the selective isolation of cell-specific primary EV. Using cardiomyocyte-derived EV (CM-EVs) as a proof-of-concept, we crossed Myh6-Cre mice with the ROSA26-mTmG reporter line to restrict membrane-targeted GFP expression to cardiomyocytes. Following myocardial infarction, cardiac EV were recovered and GFP-positive CM-EVs were selectively purified by anti-GFP nanobody-based magnetic pulldown. This approach yields highly enriched, functional cardiomyocyte-derived extracellular vesicles (CM-EVs). To accommodate diverse analytical needs, we established two distinct elution workflows: (i) a denaturing protocol optimized for downstream proteomics and immunoblotting, and (ii) a gentle, non-denaturing protocol that preserves vesicle integrity and biological activity for downstream in vitro and in vivo functional assays. Using this method, we characterized CM-EVs size distribution and proteomic signature, and demonstrated their capacity to transfer functional cargo to target cells in vitro. Because the purification approach relies solely on targeting a genetically restricted membrane tag rather than assumed surface markers, it is immediately adaptable to virtually any cell type for which a validated Cre driver exists. This method removes a long-standing bottleneck in EV research and provides a broadly applicable platform for deciphering the biochemical composition and in-vivo functions of cell-type-specific primary EV in physiological and pathological settings.
ER -