Abstract
Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.
Highlights
Extracellular vesicles (EVs) are lipid membraned nanostructures secreted by cells either directly from the plasma membrane or via the endocytic pathway [1]
Prior to EV isolation, mesenchymal stem cells (MSCs) were washed with Phosphate-buffered saline (PBS) and incubated in with EV enrichment (Exo-E) medium - containing phenol red-free low-glucose Dulbecco's modified Eagle's medium (DMEM),1% L-glutamine, 1% non-essential amino acids (NEAA), 1% penicillin/ streptomycin (P/S) and 10% Exo-free fetal bovine serum (FBS) (System Biosciences), with added DiD Vybrant Cell labelling solution according to manufacturer's instructions (5 μl/ml)
In addition to producing a variety of paracrine factors involved in tissue regeneration, MSCs produce a large number of EVs that can be collected in vitro
Summary
Extracellular vesicles (EVs) are lipid membraned nanostructures secreted by cells either directly from the plasma membrane or via the endocytic pathway [1]. Mesenchymal stem cells (MSCs) are known to be a rich source of EVs suggested to promote healing in cutaneous wounds [8], bone fractures [9] and liver injury [10]. It has recently been shown that EVs of 30 nm to 60 nm in size are more readily taken up by recipient cells within a 24-hour time period compared to larger EVs of 80 to 100 nm in size, resulting in higher motility of cells [12] These recent reports highlight the importance of size as a differentiating factor for EV populations, underlining the analysis of particle size distribution (PSD) as a crucial parameter to characterise the structural and functional properties of EVs in cell biology
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