Isolation of Extracellular Vesicles from Human Follicular Fluid: Size-Exclusion Chromatography versus Ultracentrifugation

Abstract

Follicular fluid (FF) is the microenvironment where a growing oocyte develops. Intrafollicular communication ensures oocyte competence and is carried out through paracrine signaling, the exchange of molecules via gap junctions, and the trafficking of extracellular vesicles (EVs). The study of FF-derived EVs is important for both translational and fundamental research in the female reproductive field. This study aimed to compare the efficacy and purity of two EV isolation methods: size-exclusion chromatography (SEC) and ultracentrifugation (UC). EVs isolated using SEC and UC were compared regarding their size and concentration using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA); protein contamination was assessed with microBCA; specific EV markers were detected with Western blot, and EV morphology was studied with transmission electron microscopy (TEM). Our results show that although both techniques isolated small EVs, a significantly increased yield in particle number was clear with UC compared with SEC. On the other hand, SEC generated purer EVs with fewer protein contaminants and aggregates. In conclusion, the selection of the most suited approach to isolate EVs must be conducted considering the degree of recovery, purity, and downstream application of the isolated EVs.

Profiling DNA Cargos in Single Extracellular Vesicles via Hydrogel-Based Droplet Digital Multiple Displacement Amplification

“…This can likely be attributed to SEC's ability to achieve a higher EV recovery with preserved biophysical characteristics. 45,46 To explore the potential of EV-DNA as a promising biomarker, we proceeded to evaluate its feasibility to distinguish tEVs from nEVs. Initially, EVs collected from normal hepatocyte cell lines THLE-2 and HL-7702 were found to exhibit significantly lower proportions of DNA-containing EVs than the tEVs derived from Huh-7 and HepG2 cells.…”

“…The proportion of DNA + EV in EVs obtained by SEC was slightly higher than that by UC. This can likely be attributed to SEC’s ability to achieve a higher EV recovery with preserved biophysical characteristics. , …”

Comprehensive Overview the Role of Glycosylation of Extracellular Vesicles in Cancers

“…EVs are produced by various types of cells and are consistently found in body fluids, including blood, saliva, urine, semen, breast milk, malignant ascites, and cerebrospinal fluid, as well as tissue and cell culture media. , Up to the present, researchers in relevant fields have proposed several predominant techniques for isolating EVs from complex biological samples (Figure ). These methods include ultracentrifugation (UC), differential ultracentrifugation (DC), , density gradient centrifugation (DGC), size exclusion chromatography (SEC), membrane filtration, microfluidic platforms, flow field-flow fractionation, affinity-based techniques, and immunomagnetic bead enrichment. , Among these methods, UC is regarded as the gold standard, and the combination of these techniques has been documented to yield high-quality EVs. − While each of these methods presents its own set of advantages and disadvantages, − based on the scope detailed in the present study, these isolation approaches can effectively purify EVs from complex biological samples to a certain degree. This enables both qualitative and quantitative analysis of the biomolecules carried by them.…”

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

“…Size exclusion chromatography (SEC) has been shown to be a simple and fast strategy to isolate highly pure, intact and functional EVs, with potential application in clinical setting [21][22][23] . In the present work, we aimed at optimizing a SEC protocol for the isolation of EV-AAVs, based on a protocol previously developed by our group to isolate EVs from plasma 24 .To achieve that, we took advantage of mosaic AAV vectors carrying capsid proteins from AAV1 and AAV2, thus combining the properties of both AAV serotypes that, despite their neurotropic features, are not able to efficiently cross the BBB 25 .…”