Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum

Abstract

Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the UC method in isolating exosomes from plasma, where the former contained less residual blood protein contamination. We have compared the SEC method with an optimized UC method in isolating exosomes from human serum. This was based on dilution of the serum to reduce the viscosity and a prolonged cycle of UC, followed by another four cycles. We found that >95% of serum proteins were removed without a significant loss of exosome proteins relative to SEC. We also combined one cycle of UC with SEC and found that this method provided improved results relative to the SEC method, although the serum protein contamination was several times higher than that of our optimized UC method. The TEM showed that the size distribution of exosomes isolated from each of the three methods was similar.

Keywords: SEC; UC; exosomes; serum.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.

Workflow of exosomes isolated by three different methods from human serum. Human serum was diluted with an equal volume of PBS to reduce the viscosity, followed by the centrifugation to remove cell debris and large extracellular vesicles. Next, exosomes were isolated using multiple cycles of ultracentrifugation (UC method), a commercial qEV SEC 10 mL column (SEC method), and a combination of these two methods, which first uses one cycle of UC, followed by further purification with the qEV SEC column (UC&SEC method).

Figure 2.

Protein concentration of fractions 8, 9, and 10 eluted from SEC column. In the SEC method, 0.5 mL of diluted serum was loaded onto the SEC column. The eluent was collected by fractions, where each fraction was 0.5 mL. The protein amount of each fraction was then measured using the BCA assay three times, and it was found that the ninth and tenth fractions had a much higher protein amount compared with the eighth.

Figure 3.

TEM images and the size distribution of the exosomes. TEM images showed that the exosomes isolated from the human serum by the three different methods had similar morphology and size distribution. The mean diameters of exosomes from optimized UC, UC&SEC, and SEC method were 67, 75, and 77 nm, respectively.

Figure 4.

Proteomics analysis of exosomal proteins isolated from the three isolation methods. (a) Venn diagram showed the overlap of the identified proteins from the three isolation methods. (b) Heat map of Pearson correlation coefficient shows that the proteomes of different methods had a larger variation than that of four replicates of the same method.

Figure 5.

Relative abundance of exosome markers and serum contamination protein in the samples from the three methods. (a) Relative abundance of three exosome markers (CD9, CD63, and CD81) identified in the samples from the three methods was similar. The protein amounts of CD9, CD63, and CD81 in samples from the UC&SEC column method are 1.3, 1.4, and 2.7 times that of the optimized UC method, respectively. (b) Protein amounts of ALBU, APOA1, and APOB in samples from the SEC column method are 149.2, 38.6, and 127.5 times of that of the optimized UC method, respectively.