Extracellular vesicles have been known about by scientists for many years, but it was only recently that techniques were developed to differentiate exosomes from apoptotic bodies and microvesicles.
Membrane vesicle classification and the most suitable and effective procedures for their isolation, continue to be areas of intense research. It is essential to use systems that can successfully separate vesicles when isolating them in order to prevent cross-contamination. In addition, there is a need for greater accuracy in size and concentration analysis, as well as improved workflow.
Exosomes are membrane vesicles that measure about 30 to 120nm in diameter. Released by almost every type of cell, they contain miRNA, mRNA and proteins that represent the cells secreting them.
Exosomes, which are freely available in plasma and other body fluids, have attracted a great deal of interest as therapeutic and diagnostic biomarkers. They have been shown to play a role in intercellular communication, with implications towards pro-tumor and anti-tumor activity.
Previous research has allowed an understanding of exosome isolation using density gradient ultracentrifugation, although efforts are being made to obtain more concrete confirmation of concentration and size, following purification.
This article describes a basic workflow in which centrifugation, automation and dynamic light scattering (DLS) were employed in order to purify and examine exosome samples.
The Biomek 4000 Laboratory Automation Workstation offers a reliable, high-throughput, and reproducible approach for density gradient setup, providing an efficient solution to scale-up challenges and overcome the human variable.
Preparative ultracentrifugation helps to ensure high reproducibility between runs. Notably, preparative ultracentrifugation enables the necessary g-force to be reached for timely separation of biological macromolecule samples to their isopycnic point in density gradients.
Size analysis of the fractions is made using the DelsaMax CORE DLS platform, since exosome particles can be examined in solution, with statistical significance, in less time and at less cost than with electron microscopy.
The DelsaMax CORE can analyze and size thousands of exosomes in just a single minute, rather than taking hours to examine just a few hundred particles.
Figure 1. Deck Layout of the Biomek 4000 Workstation Showing the Basic Tools Required for Gradient Prep. (1) One 24-position tube rack for placing nanotubes: the centrifuge tubes fit the existing 24-position tube rack, but new labware type had to be created to accommodate the height of the tubes; (2) one P1000 tip box for P1000 wide bore tips; (3) one Biomek 4000 Workstation P1000SL Single-Tip Pipette Tool for liquid transfer; (4) one Modular Reservoir for gradient reagents. Image credt: Beckman Coulter
Purification of exomes
Gradient preparation
A P-1000SL Single-Tip Pipette Tool and P1000 wide bore tips were used to make the density gradient on a Biomek 4000 Workstation, as shown in Figures 1 and 2. The method is flexible enough to modify volumes for individual gradients and allow changes to the number of tubes prepared.
A 24-position tube rack measuring 14mm was used to accommodate thin-wall ultracentrifuge tubes, which were programmed in as new labware.
Interface mixing of the gradients was minimized by applying a slow pipetting method with liquid level sensing. As shown in Table 1, the gradient was an overlaid gradient.
Table 1. Density Gradient
|
Gradient Layer
|
Density (g/mL)
|
Volume (mL)
|
% Iodixanol (0.25M sucrose PH7.5)
|
|
1
|
1.160
|
3
|
40
|
|
2
|
1.147
|
3
|
20
|
|
3
|
1.133
|
3
|
10
|
|
4
|
1.120
|
2
|
5
|
Differential centrifugation and density gradient run
To isolate the exosomes from Jurkat cells, cells growing in log phase were centrifuged at 750 x gfor a period of 15min to sediment the cells and this was followed by a 15min run at 2,000 x gto sediment larger debris and dead cells, using the Allegra X-15R centrifuge equipped with SX4750A rotor using sterile conical tubes (50mL).
Following this, the supernatant was centrifuged at 10,000 x g for a period of 45min at a temperature of 4°C to eliminate cellular debris and then filtered through a 0.22μm membrane before using ultracentrifugation for exosome pelleting.
The Optima XPN Ultracentrifuge and an SW 32 Ti rotor were used to pellet the exosomes at 100,000 x gfor 90min. After washing and re-suspending the exosome pellet in 1mL phosphate buffer, it was layered over the density gradient, prepared with the Biomek 4000 platform.
The Optima XPN Ultracentrifuge and SW 41 Ti rotor were used to carry out the density gradient fractionation run at 100,000 x g at a temperature of 4°C temperature for a period of 18h, with maximum acceleration and deceleration.
The tube was aliquoted into 20 fractions, with top fractions of 1mL each, 14 middle fractions of 600μl each, and the last four fractions of 400μl each. Using an Optima MAX-XP Ultracentrifuge, the resulting fractions were ultracentrifuged at 100,000 x g for a period of 1h using a TLA120.2 rotor to pellet out the exosomes.
After resuspending the pellets in 1.2mL phosphate buffer saline, the pelleting step was performed again to remove excess OptiPrep™ that could disrupt the DelsaMax CORE analysis. Finally, the pellets were resuspended in 100μl of 1x phosphate buffer saline.
Analysis of exosome size distribution
The 20 fractions were maintained at -80°C for 48h and then thawed at room temperature. The DelsaMax CORE was used to perform size analysis. 20μl of each fraction was put in DelsaMax CORE disposable cuvettes and samples were run at a temperature of 25°C temperature.
For each fraction, three measurements were taken, with each acquisition taking 20s. Phosphate buffer saline was set as the sample solvent. Following standard DLS protocols, an auto-correlation function was produced from the light scattered by the individual fractions.
The auto-correlation function was examined from 32μs to 4 * 105μs with regularization analysis. This detects multiple peaks from 1 to 10,000nm, thereby helping purity level to be determined for each fraction.
A user-defined parameter, “Fraction number” and “Density” was produced, which enabled the software to plot size against density and fraction number. The results were plotted as a % mass distribution to precisely indicate the size distribution of the biological sample.
Figure 2. Exosome Gradient Method. New tube transfer technique was created to minimize the mixing during gradient prep. Image credt: Beckman Coulter
Discussion
Figure 3 shows the size distribution results of the exosome sample preparation and indicates that density gradient is a vital step in exosome isolation. When only pelleting and ultracentrifugation, but no density gradient, are used, cellular debris and larger species are eliminated from the exosome sample.
However, small macromolecule impurities and protein remain (Figure 3A). A density gradient fractionation is needed to properly isolate the exosomes and acquire only biological particles measuring 30 to 120nm in diameter (Figure 3B).
In fractions 8–12, contamination with smaller macromolecules is eliminated, while in the first and later fractions (Figure 3B) it remains at high levels.
Figure 3. DelsaMax CORE Size Distribution (A) is the size distribution of the exosomes after ultracentrifugation without any density gradient fractionation (n=2). Note that the peak of the mass is centered between 7–9nm, indicative of high protein contamination in the exosome sample. After density gradient fractionation, (B) fractions 8–12 have their peak of mass between 60–120nm, with little evidence of protein contamination. Image credt: Beckman Coulter
Comparatively, Fractions 1–7 and Fractions 13–20 have high-protein contamination; fractions 13–20 also have contamination with larger biological macromolecules up to 500 nm in diameter.
The density of proteins is approximately 1.20g/mL, so it makes sense that there was protein contamination in the higher density fractions, (15–20). As expected, there seems to be a “sweet spot” for the density gradient; fractions 8 to 12 seem to be where exosomes migrate to during ultracentrifugation, based on density and diameter (Figure 4A).
Figure 4. Fraction Size Distribution. (A) The peak diameter for the density gradient fractions of the exosome samples stabilized between 80–120 nm at densities between 1.13 g/mL to 1.15 g/mL (Fractions 7–12), indicate later fractions vary widely in size while earlier fractions have contamination of smaller species. Exosomes have estimated densities at 1.13–1.15 g/mL.8 Data points in the orange region are most likely exosomes based on size and density. In order to avoid interference from protein contamination in the first and last fractions, the peak diameter range is from 30 nm to 1000 nm; all particles smaller than 30 nm (such as proteins) have been ignored. (B) The concentration of exosome particles also peaked between Fractions 8–12, as estimated by the amplitude of the autocorrelation function. Amplitude below 0.10, as with most fractions before fraction 8 and after fraction 12, is barely above the noise level. The low amplitude level indicates that very few particles are present and the data will be less accurate. Image credt: Beckman Coulter
As shown in Figure 4B, biological particle concentration also seems to be greatest in this fraction range, as indicated by an increase in amplitude of the autocorrelation function. The amplitude of the autocorrelation function is a measure of the total scattered light that reaches the detector.
Phosphate buffer saline and other background solvents will have amplitudes of 0.01–0.05 (a.u.). Ideal sample amplitudes range from 0.15 to 0.95, which provides sufficient signal above the background noise for precise sizing results.
It should be noted that amplitude indirectly indicates the concentration of particles; direct indication of the concentration is not possible from DLS measurements.
The potential of exosomes as therapeutic and diagnostic markers has become an important focus for researchers in this field, as has the need for reproducible and well-rounded approaches to purification.
The combination of density gradient centrifugation and differential centrifugation offers a good solution for the enrichment of exosomes from cell culture medium, while reducing the co-purification of protein aggregates and other membranous particles.
Conclusion
This article has described ways to manage the whole exosome preparation workflow, from initial centrifugation through to density gradient preparation, ultracentrifugation and final analysis.
The easy-to-use Optima MAX-XP Ultracentrifuge and Optima XPN Series Preparative Ultracentrifuge and Biomek 4000 Workstation help scientists to save time, as well as minimize errors.
The versatility and precision of the DelsaMax CORE also saves time and enhances data output, which is very valuable to researchers studying this dynamic field.
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About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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