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Standardization of Sampling for Isolation of Exosome-Like Small-Extracellular Vesicles from Peripheral Blood from Reproductive-Aged Women

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Open journal of Obstetrics and Gynecology 2018年第11期8卷 1063-1070页

作者： jéssica Maria Diehl júlia abbade tronco Natália Prearo Moco Graziela Gorete Romagnoli Ana Clara Faquineli Cavalcante Mendes de Avila juliano Coelho da Silveira Márcia Guimaraes da Silva Bruna Ribeiro de Andrade Ramos Department of Pathology Botucatu Medical School Sao Paulo State University (UNESP) Botucatu Brasil Três Lagoas Integrated College—FITL/AEMS Três Lagoas Brasil Department of Microbiology and Immunology Biosciences Institute Sao Paulo State University (UNESP) Botucatu Brasil Department of Veterinary Medicine Faculty of Zoology and Food Engineering Sao Paulo University Pirassununga Brasil

Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.

关键词： Exosome-Like Small-Extracellular Vesicles Plasma Reproductive-Aged Women

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