Božič Darja, Vrabec Katja, Železnik Ana, Raspor Andrej, Novak Valentina, Koshmak Ivana Petrović, Leskovec Maja, Štrancar Aleš
Sartorius Bia Separations, Mirce 21, SI-5270 Ajdovščina, Slovenia.
Int J Mol Sci. 2025 Aug 31;26(17):8477. doi: 10.3390/ijms26178477.
Extracellular vesicles (EVs) hold great promise in the fields of diagnostics and therapeutics. However, the heterogeneity of these membrane-enclosed messengers and the complexity of the biological samples in which they occur pose significant research challenges. The aim of this study was to improve the reliability of size-exclusion chromatography (SEC) and immunolabelling as common approaches in the EV field, and to provide a comprehensive characterisation tool for diverse EV preparations. Profiling of SEC-separated sample components was conducted through light absorbance, fluorescence, and light scattering, providing insights into particle content, size, protein content, and specific markers. Key considerations for assay robustness, including SEC mobile phase composition and immunostaining parameters, were addressed. Respecting the importance of controlled immunolabelling and preanalytical factors, the method efficiently reveals changes in the sample profile with respect to particles and small impurities. The detailed analytical capabilities and method adaptability offer a practical way to enhance the efficiency of EV research and applications.
细胞外囊泡（EVs）在诊断和治疗领域具有巨大潜力。然而，这些膜包裹的信使的异质性以及它们所在生物样品的复杂性带来了重大研究挑战。本研究的目的是提高尺寸排阻色谱法（SEC）和免疫标记作为EV领域常用方法的可靠性，并为多种EV制剂提供全面的表征工具。通过吸光度、荧光和光散射对SEC分离的样品成分进行分析，从而深入了解颗粒含量、大小、蛋白质含量和特定标志物。讨论了测定稳健性的关键考虑因素，包括SEC流动相组成和免疫染色参数。鉴于可控免疫标记和分析前因素的重要性，该方法能有效揭示样品在颗粒和小杂质方面的变化情况。详细的分析能力和方法适应性为提高EV研究和应用的效率提供了一条切实可行的途径。
