Abstract

Exosomes, nanosized extracellular vesicles (EVs) secreted by most cell types, are critical mediators of intercellular communication, carrying complex cargos of proteins, lipids, and nucleic acids that reflect the state of their parent cells. Their potential as non-invasive biomarkers for disease diagnosis and prognosis, particularly in oncology and neurodegeneration, has driven intense research interest. However, the effective and reproducible isolation of pure exosome populations from complex biological fluids, such as plasma, serum, and urine, remains a significant technical challenge. This review systematically examines the fundamental principles underlying the most common exosome isolation techniques, including ultracentrifugation, polymer-based precipitation, size-exclusion chromatography, and immunoaffinity capture. We discuss the inherent 'problems' associated with each method, such as co-isolation of non-exosomal contaminants (e.g., lipoproteins and protein aggregates), low yield, and potential alteration of exosome integrity. A comparative analysis of these methodologies highlights the trade-offs between purity, yield, and scalability. The paper concludes by emphasizing the need for standardized, high-resolution isolation protocols to unlock the full diagnostic and therapeutic potential of exosomes in membrane and cell biology research.