Shanxi Provincial Key Laboratory for Medical Molecular Cell Biology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education and Institute of Biomedical Sciences, Shanxi University, Taiyuan, China.
Center for Biomechanics and Bioengineering, Key Laboratory of Microgravity (National Microgravity Laboratory), and Beijing Key Laboratory of Engineered Construction and Mechanobiology, Institute of Mechanics, Chinese Academy of Sciences, Beijing, China.
Platelets. 2024 Dec;35(1):2337255. doi: 10.1080/09537104.2024.2337255. Epub 2024 Apr 17.
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.
外泌体携带大量蛋白质、脂质和核酸，可作为疾病诊断的多功能生物标志物和药物递送的载体。然而，目前还没有被广泛认可的用于临床应用的外泌体储存标准程序。本研究旨在确定用于血浆源性外泌体分离的最佳储存条件和抗凝剂。从健康参与者采集新鲜全血样本，并分别保存在四种不同的抗凝剂中，包括柠檬酸钠（SC1/4）、柠檬酸钠（SC1/9）、肝素锂（LH）或乙二胺四乙酸（EDTA）。通过差速超速离心从血浆中提取外泌体，并在 4°C、-20°C 或-80°C 三种不同温度下储存，储存时间从一周到六个月不等。所有用于储存条件比较的血浆样本均用 LH 抗凝剂预处理。通过透射电子显微镜、纳米颗粒跟踪分析和 Western Blotting 分别评估外泌体的形态特征、颗粒大小直径和表面蛋白谱（TSG101、CD63、CD81、CD9、CALNEXIN）。LH 和 SC1/4 组保存的外泌体倾向于保持完整的微观结构，具有丰富的蛋白质生物标志物。与-80°C 相比，4°C 短时间储存的外泌体更容易稳定。与 PBS 相比，血浆中储存的外泌体在超微结构、大小直径和表面蛋白表达方面均具有优势。总之，外泌体的特征严格取决于抗凝剂和储存温度及时间。
