Glycosylation of Extracellular Vesicles: Analytical and Translational Insights into Biomarker Discovery and Regenerative Medicine.

Glycosylation is a critical determinant of extracellular vesicle (EV) biology, shaping vesicle biogenesis, stability, biodistribution, cellular recognition, and uptake. Because EV glycans mirror disease-associated remodeling of parental cells, EV glycosylation is emerging as both a rich source of biomarkers and a functional regulator of regenerative signaling. This review highlights how altered EV glycosylation generates disease-specific signatures across major cancers, including lung, hepatocellular, colorectal, bladder, ovarian, pancreatic, and prostate cancer, and also discusses evidence in neurological, neuropsychiatric, metabolic, autoimmune, urinary, and musculoskeletal disorders. Beyond diagnostics, we examine the growing role of EV glycosylation in regenerative medicine, where glycan-dependent targeting and tissue interactions contribute to neural, cardiac, renal, skeletal, joint, and skin repair. We further provide an integrated overview of analytical strategies for EV glycosylation research, spanning mass spectrometry-based glycomics and glycoproteomics, affinity-based profiling, lectin microarrays, imaging, spectroscopic methods, advanced biosensing and nanotechnology-based approaches, and emerging artificial intelligence and bioinformatics tools. Current methodological challenges, biosafety issues, translational barriers, and future technologies are also critically discussed. Altogether, this review positions EV glycosylation as a promising interface between EV biology, precision diagnostics, and next-generation regenerative therapeutics.