Density Gradient Ultracentrifugation and Dynamic Light Scattering for High-Performance Exosome Purification and Characterization

Although researchers have been familiar with extracellular vesicles for decades, it is only recently that methods have been available for differentiating exosomes from apoptotic bodies and microvesicles.

Intensive research is still being carried out into the classification of membrane vesicles, including the most suitable and effective protocols for their isolation.  To avoid cross-contamination when isolating vesicles, it is essential to use systems that are able to separate them. There is also a need for improved workflow and increased size and concentration accuracy.

This article is focused on large-scale, cost-effective purification and fast analysis of exosomes as a way of providing solutions to these challenges. In particular, the Biomek 4000 Workstation helps to overcome human variables and offers a consistent, reproducible and high-throughput technique for gradient setup. The Optima Ultracentrifugation Series helps researchers to maintain consistency between runs and ensure that results are highly reproducible. The time and cost of TEM analysis for concentration and size is reduced by using the DelsaMax CORE.

Description of Exosomes

Almost all cell types release these exosomes or membrane vesicles, which are approximately 30 to 120nm in diameter.  They are found in body fluids such as plasma and contain mRNA, miRNA and proteins that represent the cells they are secreted from. Exosomes have become a popular focus of research, as both therapeutic and diagnostic biomarkers. They have also been shown to play a role in intercellular communication functions, with implications in both pro-tumour and anti-tumor activity.

Exosome Purification

As shown in Figures 1 and 2, the density gradient was made on a Biomek 4000 Workstation, by using a P-I000SL single-tip pipette tool and P1000 wide bore tips. The method enables the flexibility to change volumes for each gradient and also the number of tubes prepared. Thin-wall ultracentrifuge tubes (Beckman Coulter P/N 331372) were held in a 24-position, 14mm tube rack and the tubes were programmed in as new labware. Interface mixing of the gradients was reduced using a slow pipetting approach with liquid level sensing. As seen in Table 1, the gradient was an overlaid gradient.

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.

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.

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

The Allegra X- I5R centrifuge equipped with 50mL conical tubes and SX4750A rotor was used to isolate exosomes from Jurkat cells. The cells growing in log phase (final cell density of approximately 1x106 cells/mL, counted using Vi-CellXR) for 24h were centrifuged at 750 x g for 15mins to sediment the cells. This was followed by centrifugation at 2000 x g for 15mins to sediment larger debris and dead cells.

Before conducting exosome pelleting using ultracentrifugation, the supernatant was centrifuged at 10,000 x g for 45mins at 4˚C to remove cellular debris and then filtered through a 0.22µm membrane.

The Optima XPN Ultracentrifuge and SW 32 Ti rotor were used to pellet the exosomes for 90mins at 100,000 x g. The exosome pellet was washed and resuspended in 1mL of PBS (phosphate buffer saline) solution and then layered over the density gradient, prepped using the Biomek 4000 platform.  

With maximum acceleration and deceleration, a SW 41 Ti rotor and Optima XPN Ultracentrifuge were used to perform the density gradient fractionation at 100,000 x g for 18h at 4˚C. The tube was aliquoted into 20 fractions, of which the top two were 1mL each, the middle 14were 600µl each and the last four were 400µl each.

To pellet out the exosomes, ultracentrifugation of the resulting fractions was performed at 100,000 x g for 1h using an Optima MAX-XP Ultracentrifuge with a TLAI20.2 rotor. In order to remove excess OptiPrep™ (60% lodixanol), which could hinder DelsaMax CORE analysis, the pellets were resuspended in 1.2mL PBS and the pelleting step repeated. Finally, the pellets were resuspended in 100µl of 1x PBS.

Exosome Size Distribution Analysis

Prior to thawing at room temperature, the 20 fractions were frozen at -80°C for 48h. 20µl of each fraction was put in DelsaMax CORE disposable cuvettes (minimum volume, 4µl) to perform size analysis. The sample run temperature was 25°C. For each fraction, three measurements were obtained and each measurement took 20s. The sample solvent was PBS.

An auto-correlation function was generated from the light scattered by each fraction, as per DLS procedures. Using regularization analysis, the auto-correlation function was analyzed from 32µs to 4 * I05µs. Regularization analysis will detect multiple peaks from I –10,000nm, thereby aiding in the determination of the purity level of each fraction. To allow the software to plot size against density and fraction number, a user-defined parameter called “Density” and “Fraction Number” was created. In order to accurately represent the biological sample’s size distribution, results were plotted as a % mass distribution.

Exosome Gradient Method. New tube transfer technique was created to minimize the mixing during gradient prep.

Figure 2. Exosome Gradient Method. New tube transfer technique was created to minimize the mixing during gradient prep.

Results and Discussion

The importance of the density gradient as a key step in the isolation of exosomes is indicated by the size distribution results (Figure 3) of the exosome sample preparation. The use of pelleting and ultracentrifugation alone, without a density gradient, enables removal of cellular debris and larger species from the exosome sample. However, as seen in Figure 3A, small macromolecule impurities and protein still remain. As shown in Figure 3B, a density gradient fractionation is required to truly isolate exosomes and collect only biological particles of diameters ranging from 30 to 120nm.  Figure 3B also shows that contamination from smaller macromolecule is eliminated in fractions 8-12, while in the first and later fractions, it still exists at high levels.

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 7-9 nm, indicative of high protein contamination in the exosome sample. After density gradient fractionation, (B) fractions 8-12 have their peak of mass between 60-120 nm, with little evidence of protein contamination. 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.

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 7-9 nm, indicative of high protein contamination in the exosome sample. After density gradient fractionation, (B) fractions 8-12 have their peak of mass between 60-120 nm, with little evidence of protein contamination. 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.

Since the density of proteins is approximately1.20g/mL, protein contamination in the higher density fractions (15-20) is a valid concern. It can also be inferred that the density gradient has a "sweet spot". Fractions 8-12 seem to be the point to which exosomes move during ultracentrifugation, based on density and diameter, suggesting there is a “sweet spot” for the density gradient, as expected (Figure 4A).

As seen in Figure 4B, biological particle concentration also seems to be highest in this fraction range, as indicated by an increase in the amplitude of the autocorrelation function.

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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Last updated: Mar 1, 2019 at 4:33 AM

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