Comprehensive label-free characterization of extracellular vesicles and their surface proteins

Abstract

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.