Abstract
Exosomes were enriched from plasma by ultracentrifugation, precipitation, and membrane-based approaches for yield and purity. Using the four isolation approaches, particles with mode sizes within the expected range (50–200 nm) can be isolated. By protein estimation, polymer precipitation resulted in a maximum yield (5610.59 ± 51.189 µg/mL), followed by membrane affinity (471.57 ± 12.16 µg/mL), ultracentrifugation (440.22 ± 11.71 µg/mL) and filter + ultracentrifugation (235.47 ± 13.27 µg/mL). By total RNA estimation, the yield of polymer precipitation (3.26 ± 0.42 ng/mL) was higher than that of ultracentrifugation (1.52 ± 0.06 ng/mL), filter + ultracentrifugation (1.21 ± 0.25 ng/mL) and membrane affinity (1.44 ± 0.14 ng/mL). The purity of exosomal preparations was determined as the ratio of the particle number to protein and of protein to RNA. According to the ratio of the particle number to protein concentration, the “purity” of the polymer precipitation method was similar to that of the membrane affinity method and higher than that of ultracentrifugation and filter + ultracentrifugation. When the ratio of RNA to protein was used, the “purity” of the polymer precipitation method was lower than that of the membrane affinity method. Differential methods can be employed to enrich specific exosome subpopulations. The steps of the methods affect the particle number, protein content, and even exosomal purity. The best extraction and evaluation methods for exosomes need to be selected in the laboratory according to their experimental needs.
1 Introduction
Exosomes, which are used to describe a population of small extracellular vesicles (50–200 nm), have emerged as an area of intense interest owing to their important role in orchestrating intercellular communication and molecular exchange [1]. Exosomes are actively secreted by most, if not all, cells into a variety of bodily fluids (e.g., blood, saliva, urine, bronchial lavage, synovial fluid, amniotic fluid, breast milk), where the diverse array of nucleic acids, proteins, and lipids packaged within them can relay signals between the cell of origin and recipient cells [2]. The growing interest in molecules carried by exosomes as potential circulating biomarkers has prompted extensive research and the development of methods for the isolation of plasma exosomes [3]. Exosome isolation from plasma is especially difficult because of the small volume available and the technical challenge, especially due to the high density and viscosity of the sample and the complex composition of different types of vesicles, proteins, ribonucleoproteins, and lipoproteins [3].
Currently, several methodologies exist for the isolation and analysis of exosomes, such as ultracentrifugation, polymer precipitation, and membrane affinity. Several issues remain unresolved, especially in terms of clinical applications, including the aggregation of vesicles, low recovery, necessity of a large sample volume, and contamination with soluble proteins and lipoproteins [4]. Ultracentrifugation is the most common technique and is considered the “gold standard” for general exosome isolation [5]. However, this method requires special centrifugal equipment and is time-consuming. The polymer precipitation method is used to obtain exosomes by mixing the polymer with the sample and centrifuging at a low speed. This method can be used to precipitate exosomes, and solvent precipitation may influence the experimental results. The membrane affinity method is also used to extract exosomes from plasma by binding the vesicle membrane to the reagent column [3]. In addition, with recent advances in novel materials [6,7,8], exosomes are expected to be better enriched. The characterization of efficient alternative methods for exosome isolation from plasma can affect research related to biomarker discovery and clinical translation. This work aims to compare exosomes enriched from plasma by ultracentrifugation, precipitation, and membrane-based approaches in terms of yield and purity, contributing to the characterization of exosome isolation methods.
2 Materials and methods
2.1 Plasma preparation
The plasma collection and preparation protocols were approved by the Ethics Committee of Zhongshan Hospital, Xiamen University, and the participants provided written informed consent. Twenty milliliters of blood was obtained from a healthy 27-year-old volunteer in EDTA-2k anticoagulant tubes. The blood was gently mixed upside down 5 times and then centrifuged at 2,500×g for 15 min to separate plasma. Plasma was transferred to a clean tube and centrifuged again at 2,500×g for 15 min before being aliquoted, snap-frozen on dry ice, and stored at −80°C until use.
2.2 Isolation of exosomes by ultracentrifugation
In this article, we adopted two ultracentrifugation methods, ultracentrifugation, and filter + ultracentrifugation. For ultracentrifugation, 2 mL of plasma was diluted in 18 mL of ice-cold phosphate-buffered saline (PBS) and centrifuged at 10,000×g for 30 min at 4°C to pellet and remove the microvesicles. The supernatants were then centrifuged in a Beckman Coulter Optima L-100XP Ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) at 120,000×g for 2 h at 4°C with a Type 70 Ti rotor to pellet the exosomes. The supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of ice-cold PBS. A second round of ultracentrifugation (120,000×g for 2 h at 4°C with a Type 70 Ti rotor) was carried out, and the resulting exosome pellet was resuspended in 100 µL of ice-cold PBS (Figure 1).
Exosome isolation flow diagram.
For the filter + ultracentrifugation, after 2 mL of plasma was diluted in 18 mL of ice-cold PBS and centrifuged at 10,000×g for 30 min to pellet and remove the microvesicles, the supernatant was filtered using a 0.22 µm filter to remove particles larger than 0.22 µm and then subjected to ultracentrifugation as described above (Figure 1).
2.3 Isolation of exosomes by polymer precipitation
For polymer precipitation, 2 mL of plasma was centrifuged at 10,000×g for 30 min at 4°C to pellet and remove microvesicles. The supernatant was added to a 15 mL centrifuge tube, followed by a precipitation procedure. The supernatant was mixed with 2 mL of Blood PureExo Solution (Umibio, Shanghai, China) and 8 mL of cold PBS, incubated at 4°C for 2 h, and then centrifuged at 10,000×g for 1 h. The resulting supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 0.8 mL of PBS. A second round of centrifugation (12,000×g for 2 min at 4°C) was carried out, and the resulting exosome supernatant was transferred to the upper part of the Exosome Purification Filter column (Umibio, Shanghai, China) and centrifuged at 3,000×g for 10 min. The eluate of the exosome column was collected for the following analysis (Figure 1).
2.4 Isolation of exosomes by the membrane affinity method
Two milliliters of plasma was centrifuged at 10,000×g for 30 min at 4°C to pellet and remove microvesicles. The supernatant was carefully collected and mixed 1:1 with buffer XBP (Qiagen, Hilden, Germany) and then added to an exoEasy spin column. After centrifugation at 500×g for 1 min, the flowthrough was discarded, and 10 mL of wash buffer was added to the column to wash away nonspecifically retained material. After another centrifugation (5,000×g for 5 min) and elimination of the flowthrough, the eluate with exosomes was collected by adding 400 µL of Buffer XE to the spin column and incubated for 1 min. After centrifugation at 500×g for 5 min, the eluate from the exosome column was collected for the following analysis (Figure 1).
2.5 Transmission electron microscopy
Exosomes were visualized using transmission electron microscopy as previously described, with slight modifications [9]. Briefly, exosome suspensions were diluted 1:50 with PBS, and 20 µL of exosome preparations was allowed to adsorb on a copper mesh for 3 min and was negatively stained with 2% (w/v) phosphotungstic acid for 5 min. Transmission electron microscopy analysis was performed at an acceleration voltage of 80 kV with a transmission electron microscope (H-7650; Hitachi, Ltd, Tokyo, Japan).
2.6 Protein quantification
Protein concentration was detected using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer's instructions.
2.7 Western blot analysis
The samples were mixed with 5× loading buffer and heated at 95°C for 5 min. Volumes corresponding to 25 mg of protein from isolates were separated on a 5–12% polyacrylamide gel. Samples were then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories), which was blocked with 5% nonfat milk in Tris-buffered saline (TBS) for 1 h. The membrane was incubated with primary antibodies against calnexin (1:1,000; Abcam), albumin (1:500; Abcam), TSG101 (1:500; Abcam), galectin-3 BP (1:500; Abcam), and CD9 (1:1,000; Abcam) dissolved in 0.05% TBS-Tween overnight at 4°C, and then, the membrane was washed with 0.05% TBS-Tween for 10 min three times. Secondary antibodies were diluted in 0.05% nonfat milk in TBST and incubated for 1 h. The membrane was analyzed with ECL Prime Western Blotting Detection.
2.8 Nanoparticle tracking analysis
Exosome concentration was analyzed using a NanoSight LM10 system (NanoSight Ltd, Navato, CA) equipped with a blue laser (405 nm). Nanoparticles were illuminated by the laser, and their movement under Brownian motion was captured for 60 s. The process was repeated three times. Then, all three recorded videos were subjected to nanoparticle tracking analysis (NTA) using NanoSight particle tracking software to calculate exosome concentrations and size distribution.
2.9 Total RNA extraction
Total RNA was isolated using a miRNA Serum/Plasma Advanced Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The RNA concentration and integrity were determined by capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Rockville, MD). For the detection of mRNA in exosomes, the total isolated RNA was converted to cDNA using a PrimeScriptTM RT reagent Kit (TaKaRa Bio, Inc., Kusatsu, Japan) and oligo (dT) primers. cDNA detection was performed using a Bioanalyzer. Quantification of miR-130 was performed using miR-130-specific forward primers and universal reverse primers.
2.10 Statistics
Data are expressed as the mean ± SD and were assessed using SPSS 18.0 statistical software (SPSS, Inc., Chicago, USA). One-way analysis of variance (ANOVA) was used to assess the differences between multiple groups, followed by Tukey’s post hoc test. A P value <0.05 was considered significant.
3 Results
3.1 Morphological characterization of exosomes extracted by different isolation procedures
Transmission electron microscopy analysis revealed that all isolation procedures successfully isolated exosomes with the expected size range and morphology. Cup-shaped vesicles were observed with heterogeneous sizes ranging from approximately 50–200 nm (Figure 2). The morphology of exosomes in samples was not different in the ultracentrifugation, filter + ultracentrifugation, and membrane affinity groups, and in the polymer precipitation group, the shape of exosomes was slightly irregular.
Electron microscopic image of exosomes following different isolation methods. Morphology of exosomes (white arrow indicate) isolated using (a) ultracentrifugation, (b) filter + ultracentrifugation, (c) membrane affinity, (d) and polymer precipitation. Cup-shaped vesicles with a heterogeneous size (50–200 nm diameter) were clearly visualized in all preparations. The bars indicate 100 nm.
3.2 The size and concentration of exosomes extracted with different isolation procedures
NTA profiles of exosomes obtained with the different isolation procedures revealed that the mode size was in the expected range of 50–200 nm. The major peaks of the ultracentrifugation, filter + ultracentrifugation, polymer precipitation, and membrane affinity groups were 96.6 ± 2.7, 97.9 ± 0.6, 96.6 ± 0.9, and 97.5 ± 0.5 nm, respectively (Figure 3a and b). In the exosomes enriched by membrane affinity, we observed several subpopulations of differently sized vesicles with multiple concentration peaks in NTA. NTA quantification confirmed that polymer precipitation resulted in the highest number of particles (6.20 ± 1.74 × 1011), followed by the membrane affinity (6.06 ± 1.90 × 1010), ultracentrifugation (5.86 ± 0.72 × 1010), and ultracentrifugation + filter methods (1.60 ± 0.40 × 1010). The number of enriched particles was not different among the membrane affinity, ultracentrifugation, and ultracentrifugation + filter groups (Figure 3c).
Size distribution and concentration of exosomes enriched with different isolation procedures. (a) Particle size for plasma exosomes measured by nanoparticle tracking analyses (NTA). Insets depict higher magnifications of the black dot box areas. (b) Size distribution as a percentage of vesicles larger than 195 nm (black), between 35 and 195 nm (gray) and smaller than 35 nm (white) based on the same data as in (a). (c) Particle concentration of exosomes extracted by different isolation procedures. The results are the mean ± standard error of the mean of three independent experiments. One-way analysis of variance (ANOVA) was used to assess the differences between multiple groups, followed by Tukey’s post hoc test. **P < 0.01. NS, P > 0.05.
3.3 The yield of exosomes from different isolation procedures
The total exosome yield was determined by protein estimation from intact exosomes using the BCA protein assay. We observed that the polymer precipitation method had the maximum yield (5610.59 ± 51.189 µg/mL), followed by the membrane affinity (471.57 ± 12.16 µg/mL), ultracentrifugation (440.22 ± 11.71 µg/mL), and filter + ultracentrifugation methods (235.47 ± 13.27 µg/mL). The filter + ultracentrifugation method yielded the lowest level of exosomes (Figure 4a).
The yield of exosomes from different isolation procedures. (a) The exosomal yield presented as the protein estimation. (b) The exosomal yield presented as the total RNA. (c) Representative bioanalyzer profiles of RNA analyzed by an Agilent 2100 Bioanalyzer; the y-axis shows fluorescence units (FU), and the x-axis shows the nucleotide length (nt) of the RNA. The results are the mean ± standard error of the mean of three independent experiments. One-way analysis of variance (ANOVA) was used to assess the differences among multiple groups, followed by Tukey’s post hoc test. **, P < 0.01; ***, P < 0.001. NS, P > 0.05.
As exosomes are considered an important source of RNA-based biomarkers, we also assessed exosome yield based on the total RNA content of exosomes determined by a Bioanalyzer. We found that RNA recovery by polymer precipitation (3.26 ± 0.42 ng/mL) was higher than that of ultracentrifugation (1.52 ± 0.06 ng/mL), filter + ultracentrifugation (1.21 ± 0.25 ng/mL), and membrane affinity (1.44 ± 0.14 ng/mL), and the RNA recovery by the latter three methods was not significantly different (P > 0.05) (Figure 4b). The Bioanalyzer profiles of isolated RNA showed the presence of small RNAs for different isolation procedures (Figure 4c).
In addition, to evaluate the yield of exosomes obtained from each isolation procedure, we performed an immunoblot with the loading of equal amounts of protein and demonstrated that the expression of Galectin-3 BP, CD9, and Tsg101 – all considered late endosomal markers enriched in exosomes – was positive in each isolation procedure. The expression levels of Galectin-3 BP, CD9, and Tsg101 were different in isolation procedures under the same protein loading. Galectin 3 BP was expressed at the lowest level in the polymer precipitation group, and TSG101 and CD9 were both expressed at the lowest level in the membrane affinity group (Figure 5).
Surface marker expression profiles in exosome preparations. (a) Western blot for Galectin-3 BP, Tsg101, and CD9 expression in each exosome preparation (20 µL of total exosomal protein was loaded). (b) Quantification of the expression levels of Galectin-3 BP, Tsg101, and CD9 in each exosome preparation. The results are the mean ± SE of the mean of three independent experiments. One-way analysis of variance (ANOVA) was used to assess the differences among multiple groups, followed by Tukey’s post hoc test. * P < 0.05, ***P < 0.001.
3.4 The purity of exosomes extracted by different isolation procedures
The purity of the exosomes was determined by the ratio between particle concentration and protein. Experiments showed that exosome extraction by membrane affinity and polymer precipitation showed excellent purity [(130.83 ± 43.50) × 107 vs (110.41 ± 30.47) × 107 particles/µg, P > 0.05], while ultracentrifugation [(13.36 ± 1.70) × 107 particles/µg] and filter + ultracentrifugation [(7.04 ± 2.22) × 107 particles/µg] resulted in the lowest purity (Figure 6a).
Purity of exosomes from different isolation procedures. (a) The exosomal purity presented as a ratio between particle concentration and protein. (b) Exosomal purity presented as the RNA (µg) to protein (µg) ratio. (c) Western blots for calnexin and albumin expression in each exosome preparation (20 µL of total exosomal protein was loaded). The results are the mean ± SE of the mean of three independent experiments. One-way analysis of variance (ANOVA) was used to assess the differences among multiple groups, followed by Tukey’s post hoc test. *, P < 0.05. **, P < 0.01. NS, P > 0.05.
The ratio of RNA to protein was previously found to be higher in exosomes than in microvesicles, suggesting that more RNA is associated with exosomes [10]. We also calculated the RNA to protein ratio to determine the exosomal purity. This ratio was significantly lower for the exosomes isolated by polymer precipitation than for the exosomes isolated by ultracentrifugation, filter + ultracentrifugation, or membrane affinity (Figure 6b). There was no difference in the ratio of RNA to protein among the latter three groups.
As plasma is a very complex and viscous fluid, the presence of contaminants is another method to determine the purity of exosomes. We assessed calnexin and albumin contamination by Western blotting. All samples were negative for calnexin, an endoplasmic reticulum component protein. Albumin was present in all exosome extracts analyzed, and the membrane affinity group had the highest albumin contamination (Figure 6c).
3.5 Practicability of different isolation procedures
For the practicability of different isolation procedures, ultracentrifugation requires special equipment (ultracentrifuge) and takes approximately 4 h. Special training is needed for researchers who are learning to perform exosome extraction by applying the ultracentrifugation method. After simple training, nonprofessionals can perform and master the membrane affinity and polymer precipitation approaches. The isolation time was approximately 3 h 12 min for polymer precipitation and only 25 min for membrane affinity (Table S1).
4 Discussion
Exosomes have emerged as a promising alternative source of biomarkers for several diseases since they can be obtained from almost all biological fluids [2]. As a result, efficient isolation of exosomes has been an active area of research to understand their biological properties and to explore their potential in biomarker development for early disease diagnosis [11]. In this article, we evaluated four exosome isolation methods and generated significant data on their relative efficacy with regard to exosome yield and purity. The NTA results showed that the diameters of the exosomes extracted by the four methods all met the standard exosome size of 50–200 nm. Among all the methods tested, the polymer precipitation method had the maximum yield (5610.59 ± 51.18 µg/mL), followed by membrane affinity (471.57 ± 12.16 µg/mL), ultracentrifugation (440.22 ± 11.71 µg/mL), and filter + ultracentrifugation (235.47 ± 13.27 µg/mL), as shown by protein estimation. Patel et al. also found that the precipitation-based method yielded the highest quantity [12], and the highest yield in the precipitation method could be likely due to precipitation of extracellular vesicles other than exosomes or additional aggregated proteins. The polymer precipitation method also provided the most particles and the maximum yield of total RNA. Differential protein, particle, or total RNA yields reflected different exosome quantities obtained from various methods.
Exosomes are nanometer-sized (50–200 nm) extracellular vesicles that are actively shed from cells into body fluids. In this article, the plasma was filtered using 0.22 µm filters to remove particles larger than 0.22 µm [13]. We accordingly investigated the purity and yield of exosomes extracted by ultracentrifugation and filter + ultracentrifugation. The protein concentration of the exosomes was low in the filter + ultracentrifugation group, but the number of particles and the total RNA yield did not decrease when using 0.22 µm filters. Therefore, we believe that although the diameter of the exosomes is less than 200 nm, a filter membrane with an aperture of 0.22 µm can still absorb a certain amount of exosomes, which may be related to the protein concentration of the exosome extract obtained by filter + ultracentrifugation.
Purity is an indispensable index for exosome evaluation. As previously reported, the purity of exosomal preparations was determined as a ratio of particle number to protein concentration [14,15] or a ratio of RNA to protein concentration [3]. Interestingly, for the membrane affinity method, the results of the two calculation methods were completely opposite. According to the ratio of the particle number to protein concentration, the “purity” of the polymer precipitation method was similar to that of the membrane affinity methods and higher than that of the ultracentrifugation and filter + ultracentrifugation methods. When the ratio of RNA to protein concentration was used, the “purity” of the polymer precipitation was lower than that of the membrane affinity method. Using different purity evaluation methods, we found that the exosomal preparations showed different levels of “purity”; thus, researchers need to perform appropriate exosome purity evaluations according to the experimental purpose.
As plasma is a complex and viscous fluid with a protein concentration of approximately 60–80 g/L, the isolation of exosomes to measure proteins that are in the range of 10−6 g/L is a major challenge [16,17]. For this reason, it is important not only to have a method that can purify exosomes efficiently but also have a method that can remove most impurities from plasma [4]. In this article, we did not detect contaminant debris from the endoplasmic reticulum marker calnexin, an endoplasmic reticulum component, thus eliminating the presence of cell contamination in the exosomal preparations. However, another serum contaminant, albumin, was present in all exosome extracts analyzed, and the membrane affinity method had the highest albumin level. The effect of albumin contamination on the function of extracted exosomes needs to be further evaluated. In addition, we did not assess the relevant marker of lipoprotein, which needs to be assessed in future studies.
Ultracentrifugation has been the most widely used method for exosome extraction but there are some limitations in the application of this method [18]. This strategy requires special equipment and takes a long time (approximately 4 h), and special training is needed for researchers who are learning to perform exosome extraction by applying the ultracentrifugation method [5]. The polymer precipitation method and membrane affinity method can effectively shorten the required time but albumin can be either nonspecifically bound to exosomes or obtained during the process of purification, which was confirmed by the polymer precipitation method and membrane affinity method showing higher albumin contamination than ultracentrifugation [9]. How to balance the ease of operation and albumin contamination is an urgent problem to be solved in the clinical application of plasma exosomes.
To uniformly compare the extraction efficiency of exosomes by different methods, we only used single samples for comparison but almost all cells could produce exosomes, and the interindividual variability could be high. A multiperson sample comparison of special samples (hemolytic, lipemic, or icteric samples) is also necessary.
5 Conclusion
Current research on exosomes is challenged by efficient isolation strategies with high specificity that are practicable. This study suggests that different methods can be employed to enrich specific exosome subpopulations. Our results also indicate that the exact steps of differential methods affect the particle number, protein content, and even purity of exosomes. The laboratory needs to select the best extraction and evaluation methods of exosomes according to their experimental needs.
Acknowledgements
This work was supported by the National Natural Science Foundation of China [grant numbers 82172331, 82102514, 81972028, 81871729, and 81672094] and the Key Projects for Province Science and Technology Program of Fujian Province, China [grant numbers 2020D017 and 2019D008]. The funders played no role in the study design, data collection, analyses, decision to publish, or manuscript preparation.
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Author contributions: WJ Li, H Chen, and L-R Lin conceived and designed the experiments. J-J Niu and X-Z Zhu performed the experiments. M-L Tong analyzed the data. L-R Lin wrote the paper.
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Conflict of interest: The authors of this article have no conflicts of interest to disclose.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
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