Browsing by Subject "Size-exclusion chromatography"

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    A Size-Exclusion Chromatography-Based Procedure for Isolating Extracellular Vesicle Subsets from Porcine Seminal Plasma
    (Humana Press, 2024-04-10) Martínez Díaz, Pablo; Ana Parra; Christian M Sanchez-López; Antonio Marcilla; Bucci, Diego; Roca Aleu, Jorge; Barranco Cascales, Isabel; Medicina y Cirugía Animal; Facultad de Veterinaria
    Extracellular vesicles (EVs), membrane nanoparticles (30-to-1000 nm diameter) secreted and released by most of the body functional cells, have emerged as powerful cell-to-cell messengers transferring their bioactive cargo (proteins, lipids, and nucleic acids) from donor to recipient cells. The promising potential utility of EVs as both noninvasive biomarkers and therapeutic carriers for several pathologies, including some types of cancers, has attracted increasing scientific interest. EVs can be found in all body biofluids, including seminal plasma, a complex fluid consisting mainly of a mixture of secretions of the epididymis and accessory sex glands. Seminal EVs are involved in modulating both sperm physiological processes and immune environment of the internal female genital tract, thus playing an essential indirect role in fertilization and embryo development. Seminal plasma, alike other biofluids, contains a heterogenous population of EV-subsets. However, the lack of consensus on the most accurate procedure for isolating EV-subsets has led to a poor definition of their composition/function. Currently, size exclusion chromatography (SEC), a size-selective separation method, is one of the most promising EV-isolation procedures, allowing the isolation of EVs from biological fluids in a purer, easier, cheaper, and more scalable way compared to other alternative isolation procedures. This chapter reports a SEC-based protocol, combined with differential centrifugation and ultrafiltration, to isolate two subsets of seminal EVs differing in size (large and small EVs) in the ejaculate of pigs, a livestock species of great productive interest and an outstanding animal model for human reproduction.
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    Isolation and characterization of extracellular vesicle subsets in donkey seminal plasma
    (Elsevier, 2025-05-22) Catalán, Jaime; Martínez Díaz, Pablo; Parra, Ana; Bonet, Sergi; Yeste, Marc; Roca, Jordi; Barranco Cascales, Isabel; Miró, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
    Seminal plasma (SP), a fluid composed of secretions from the male genital tract, is rich in seminal extracellular vesicles (sEVs), nano-sized particles surrounded by a lipid bilayer membrane and loaded with functionally active molecules. Seminal EVs are secreted by functional cells of the male genital tract and play a key role in modulating reproductive processes, including sperm function and immune response in the female genital tract. The aim of this study was to isolate and characterize sEVs from donkey SP for the first time. Nine SP samples were collected from nine healthy and reproductive active donkeys. The SP samples were randomly pooled to create three pools (three SP samples per pool). The SP pools were subjected to differential centrifugation and size-exclusion chromatography to separately isolate two subsets of sEVs: small (S-) and large (L-). Orthogonal characterization of sEV samples was performed according to MISEV 2023 guidelines, including morphology (by cryogenic electron microscopy), concentration (by total protein concentration and total and CFSE positive particles by flow cytometry [FC]), particle size distribution (by dynamic light scattering), purity (by albumin assessment by FC), and specific EV protein markers (tetraspanins CD9, CD63, and CD81, and HSP70 by FC). The results showed that donkey SP is highly enriched in sEVs. Size differences were found between both sEV subsets, with S-sEVs being smaller (∼160 nm) and L-sEVs larger (∼290 nm). Both sEV subsets were positive for the four EV protein markers. However, the percentage of CD81-positive events was higher in S-sEV samples than in L-sEV samples (P < 0.05). This study is the first to isolate and characterize sEVs in donkey SP, demonstrating their heterogeneity and suggesting differences in biogenesis and function between S-sEVs and L-sEVs.