Comparison of ultracentrifugation and density gradient separation methods for isolating Tca8113 human tongue cancer cell line-derived exosomes

Abstract

The aim of the present study was to compare the method of ultracentrifugation and density gradient separation for isolating Tca8113 human tongue squamous cell carcinoma cell line-derived exosomes. The exosomes were obtained from the culture supernatant of cultured Tca8113 cells, respectively, followed by identification with transmission electron microscopy observation and western blot analysis. The two different methods were then compared by the morphology, the distribution range of the particle size and the concentration of proteins of the extracted exosomes. In vitro, Tca8113 cells can secrete a large amount of vesicle-like structures, which are identified as exosomes by the presence of the surface markers, Hsp-70 and Alix. The protein profile of the two products are almost the same, however the particle size distribution of the exosomes extracted with density gradient centrifugation are more limited, between 40–120 nm, and these have a higher protein concentration. The results indicate that Tca8113 cells can secrete exosomes in vitro, and the density gradient separation methods for purifying exosomes is improved, which is helpful for future research and application of exosomes.

Size-dependent cellular uptake of exosomes

“…UC exosomes from saliva were found to be smaller than EQ as well as different in morphology and protein content [18]. UC exosomes isolated from cancer cell supernatants were also found larger than those purified by density gradient from the same source [35]. Density gradient was evaluated as the best method to study exosomes RNA, although the method is characterized by lower yields when compared to UC and EQ [9].…”

Symposium review: The relevance of bovine milk phospholipids in human nutrition—Evidence of the effect on infant gut and brain development

“…In contrast with the vast number of publications regarding structure and biological effects of the MFGM, a new group of vesicular structures found in milk are getting more and more attention for their potential effects on human health. Milk exosomes are cell-released phospholipid vesicles ranging from 30 to 120 nm in diameter and with a density of 1.13 to 1.19 g/mL (Zhang et al, 2014) that enclose a variety of cargo, including mRNA, microRNA, long noncoding RNA, DNA, and proteins. In milk, both human and bovine, MEX have been shown to transfer their RNA-derived cargo to immune cells to potentially modulate immune cell function (Lakkaraju and Rodriguez-Boulan, 2008;Hata et al, 2010;Zempleni et al, 2017).…”

Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices

“…In density-gradient separation, a sample is spun in a tube that contains a density gradient of a viscous material, such that objects separate based on their isopycnic point. 57 While this technique can achieve higher purity and recovery rate than conventional differential centrifugation, 60 it cannot separate exosomes from viruses or microvesicles due to their similar buoyant density. 57 The run times for density gradient separation are similar to conventional ultracentrifugation, and requires the same ultracentrifugation equipment, making it impractical for many clinical applications.…”