Preservation of Extracellular Vesicle Integrity and Cargo Using Spiral Microfluidic Chip‐Based Separation

ABSTRACT Extracellular vesicles (EVs) are lipid bilayer–enclosed nanoparticles that carry molecular cargos, such as miRNAs and membrane proteins, and hold promise as minimally invasive biomarkers. However, their potential remains limited by separation methods, which can degrade microRNA (miRNA) cargo and compromise vesicle integrity. Among these approaches, ultrafiltration (UF) is commonly used but prone to inducing EV cargo loss and deformation. In contrast, a microfluidic chip (MF) has emerged as a more efficient alternative, preserving cargo integrity and vesicle structure. In this study, a spiral MF is evaluated to preserve EV integrity. EVs separated with MF (MEVs) show a broader repertoire of cancer‐related miRNAs compared to those separated with UF (UEVs). These miRNAs are significantly enriched in pathways, such as PD‐L1 signaling and non‐small cell lung cancer. Cryogenic transmission electron microscopy analysis confirms that MEVs maintain intact bilayer structures, while UEVs often display deformation and membrane loss. Sandwich enzyme‐linked immunosorbent assays show higher expression of general and cancer‐related biomarkers, including PD‐L1, CEA, CA125, EpCAM, and CD63 in MEVs. Additionally, MF requires less processing time and smaller volumes than UF. Overall, a spiral MF provides a reliable EV separation platform that maintains miRNA cargo and membrane integrity, supporting accurate analyses and diagnostics.