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

Autor: Soares M; Institute for Interdisciplinary Research (IIIUC), University of Coimbra, 3030-789 Coimbra, Portugal.; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal., Pinto MM; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-548 Coimbra, Portugal., Nobre RJ; Institute for Interdisciplinary Research (IIIUC), University of Coimbra, 3030-789 Coimbra, Portugal.; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; ViraVector, University of Coimbra, 3004-504 Coimbra, Portugal., de Almeida LP; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-548 Coimbra, Portugal.; ViraVector, University of Coimbra, 3004-504 Coimbra, Portugal., da Graça Rasteiro M; CIEPQPF, Department of Chemical Engineering, University of Coimbra, Pólo II, R. Silvio Lima, 3030-790 Coimbra, Portugal., Almeida-Santos T; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; Reproductive Medicine Unit, Centro Hospitalar e Universitário de Coimbra, Praceta, R. Prof. Mota Pinto, 3004-561 Coimbra, Portugal.; Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-548 Coimbra, Portugal., Ramalho-Santos J; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal., Sousa AP; CNC-Center for Neuroscience and Cell Biology, CIBB, University of Coimbra, Azinhaga de Santa Comba, Celas, 3004-504 Coimbra, Portugal.; Reproductive Medicine Unit, Centro Hospitalar e Universitário de Coimbra, Praceta, R. Prof. Mota Pinto, 3004-561 Coimbra, Portugal.
Jazyk: angličtina
Zdroj: Biomolecules [Biomolecules] 2023 Feb 02; Vol. 13 (2). Date of Electronic Publication: 2023 Feb 02.
DOI: 10.3390/biom13020278
Abstrakt: 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.
Databáze: MEDLINE
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