Isolation of exosomes from serum of patients with lung cancer: a comparison of the ultra-high speed centrifugation and precipitation methods.

Abstract

BackgroundExtracellular vesicles (EVs) is a group of heterogeneous cell-derived membrane structures, which is composed of a large number of exosomes released by cells, microbubbles (MVs) and apoptotic bodies. The formation of exocrine body is a process of fine regulation, which includes four stages: initiation, endocytosis, polycystic body formation and exocrine body secretion. Ultracentrifugation is currently the gold standard for external body separation; it includes a series of centrifugation steps at a rotation speed of 100,000 rpm or more to purify exocrine bodies from protein contaminants. Thus far, some in vitro separation methods, such as ultracentrifugation, polymer-based exosome separation kits and immune affinity-based isolation using antibodies against exosome surface proteins, have been used for tumor exosome isolation. It is not very clear which method is more suitable for the separation of serum exosomes from lung cancer patients.MethodsTwo methods for the extraction of exosomes from serum samples of lung cancer patients, namely, ultra-high speed centrifugation (Ultra-Exo) and precipitation (Prekit-Exo), were analyzed and compared. The biological morphologies of the extracted exosomes were studied by negative staining matter with transmission electron microscopy and cryo-electron microscopy. The particle size and the distribution were detected using nanoparticle tracking analysis (NTA).ResultsBio-transmission electron microscopy revealed that the morphologies of exosomes extracted by ultracentrifugation were superior to exosomes extracted with the Prekit-Exo kit. Ultracentrifugation was able to extract more exosomes compared to the Prekit-Exo kit. NTA showed that the exosomes obtained by ultra-high speed centrifugation had a smaller particle size compared to exosomes obtained by precipitation (30.4±26.8 vs. 150.3±6.8 nm, respectively). It is possible that the precipitant used in the precipitation kit was extracted with the exosomes, thereby causing the particle size to increase. Notably, the particle size of the exosomes extracted by the precipitation kit method showed a relatively narrow range in size. This could be due to the coating effect of the precipitation reagent, reducing the difference in the particle size of the exosomes.ConclusionsExosomes collected from the serum of lung cancer patients using the two extraction methods differed in morphology and numbers, with the ultracentrifugation method being superior to the precipitation method.