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

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-der...

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Bibliographic Details
Published in:Biomolecules (Basel, Switzerland) Switzerland), 2023-02, Vol.13 (2), p.278
Main Authors: Soares, Maria, Pinto, Maria M, Nobre, Rui Jorge, de Almeida, Luís Pereira, da Graça Rasteiro, Maria, Almeida-Santos, Teresa, Ramalho-Santos, João, Sousa, Ana Paula
Format: Article
Language:English
Subjects:
Centrifuges
Chromatography
Chromatography, Gel
Comparative analysis
Contaminants
Contamination
Extracellular matrix
Extracellular vesicles
Extracellular Vesicles - metabolism
Female
Follicular Fluid
Gap junctions
Humans
Light scattering
Methods
Microenvironments
MicroRNAs
Nanoparticles
Oocytes
Ovaries
Paracrine signalling
Physiological aspects
Polycystic ovary syndrome
Proteins
Proteins - metabolism
size-exclusion chromatography
Transmission electron microscopy
Ultracentrifugation
Ultracentrifugation - methods
Ultrasonic imaging
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Summary: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.
ISSN:2218-273X
2218-273X
DOI:10.3390/biom13020278