Storage of plasma-derived exosomes: evaluation of anticoagulant use and preserving temperatures

Exosomes carry large cargo of proteins, lipids, and nucleic acids, serving as versatile biomarkers for disease diagnosis and vehicles for drug delivery. However, up to date, no well recognized standard procedures for exosome storage were available for clinical application. This study aimed to determine the optimal storage conditions and the anticoagulants for plasma-derived exosome isolation. Fresh whole blood samples were collected from healthy participants and preserved in four different anticoagulants including sodium citrate (SC1/4), sodium citrate (SC1/9), lithium heparin (LH), or Ethylenediamine tetraacetic acid (EDTA), respectively. Exosomes were extracted from the plasma by differential ultracentrifugation and stored at three different temperatures, 4°C, -20°C or - 80°C for a duration ranging from one week to six months. All plasma samples for storage conditions comparison were pretreated with LH anticoagulant. Exosome features including morphological characteristics, pariticles size diameter, and surface protein profiles (TSG101, CD63, CD81, CD9, CALNEXIN) were assessed by transmission electron microscopy, Nanoparticle Tracking Analysis, and Western Blotting, respectively. Exosomes preserved in LH and SC1/4 group tended to remain intact microstructure with highly abundant protein biomarkers. Exosomes stored at 4°C for short time were prone to be more stable compared to thos at -80°C. Exosomes stored in plasma were superior in terms of ultrastructure, size diameter and surface protein expression to those stored in PBS. In conclusion, plasma-dervied exosome characteristics strictly depend on the anticoagulants and storage temperature and duration.What is the context? Effective isolation of exosomes is a prerequisite for subsequent investigation into its involvemnt in disease development as well as potentialtherapeutic applications.Anticoagulants, storage temperature and durations might change the microscopical structure, integrity and also the stability of plasma-derived exosomes. However, no internationally recognized standard of exosome storage procedure was available for clinical use.What is new? Our finding evaluated the effect of anticoagulants and storage on plasma exosome characteristics.Exosomes isolated from plasma preserved with Li-heparin and sodium citrate (1/4) showed better physical properties and surface marker protein expression.Isolated exosomes appeared more stable in a short time for 4°C compared to −80°C. Storage of exosomes in plasma showed better physical properties and surface marker protein expression than in PBS.What is the impact? Our findings inform the significance of standardizing procedure of exosome isolation and preservation.