Comparative Evaluation of Methods for Isolating Small Extracellular Vesicles Derived from Pancreatic Cells

Abstract

Background: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation have been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs derived from pancreatic cancer cells.Results: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. For the concentration process, ultracentrifugation method obtained high quality and concentration pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs. Conclusions: For isolating sEVs derived from pancreatic cancer cells, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be suitable for isolation of sEVs for therapeutic study, immunoaffinity capturing may be applied for studies exploring sEVs as biomarkers.