Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Extracellular vesicles or EVs are nano-sized, heterogenous, membrane-bound vesicles secreted by various cells, including brain tumor cells. The small EVs are a subpopulation comprising endosomal-derived and membrane-shed EVs.

To purify the small EVs, begin by taking pre-extracted EVs suspended in a sucrose supplemented buffer that maintains a homogenous solution and preserves the structural integrity of EVs. 

To this solution, then, add an appropriate volume of iodixanol - an inert and non-toxic density gradient medium - to achieve the desired density.

Iodixanol solubilizes the contaminating proteins to be removed later.

Next, add layers of iodixanol solutions of successively decreasing densities to create a discontinuous density gradient.

Centrifuge the tube at high speed.

Depending upon their floatation rates, the denser EVs migrate to an equally-dense layer of gradient medium while less-dense EVs migrate further to a lighter layer, and the densest contaminants form a pellet at the bottom of the tube.

Now, transfer small fractions of the gradient layers in fresh tubes containing a suitable buffer.

Thereafter, centrifuge the tubes at ultra-high speed in chilled conditions.

During centrifugation, low temperature prevents EVs from high-speed-induced heat damage.

After the pure EVs pellet down, remove the supernatant. Resuspend the EVs in an appropriate buffer and proceed for characterization and proteomics analysis.

First, add 1.5 milliliters of 60% iodixanol to the 1.5 milliliters of the sucrose/Tris buffer that contains the EVs to create a final solution containing 30% iodixanol. Pipette up and down several times to mix the solution thoroughly.

Transfer this solution to the bottom of a 5.5-milliliter ultracentrifugation tube. Next, mix the 60% iodixanol stock with ultrapure water to prepare at least 1.5 milliliters of both a 20% and a 10% iodixanol solution.

Using a syringe and an 18G needle, measure 1.3 milliliters of the 20% iodixanol solution and carefully layer it on top of the bottom gradient.

Keep the bevel of the needle in contact with the inside of the tube just above the meniscus and add the solution dropwise to avoid mixing the layers at the density interface.

Then, layer 1.2 milliliters of the 10% iodixanol solution on top of the 20% layer using the same technique. Carefully balance and load the ultracentrifugation tubes into rotor buckets.

Set the acceleration and deceleration speeds of a swing bucket rotor to the minimum rates and centrifuge at 268,000 x g and at 4 degrees Celsius for 50 minutes.

While the sample is being centrifuged, label ten 1.5-milliliter microcentrifuge tubes for each sample that will correspond with fractions 1 through 10 of the density gradient.

Once the centrifugation is complete, gently remove the tubes from the rotor buckets and place them into a stable holder. Pipette 10 serial fractions of 490 microliters from the top of the gradient into the corresponding tubes.

Using a refractometer, measure the refractive indices of the fractions. Then, transfer each fraction to a clean 12-milliliter ultracentrifugation tube.

Add 5 milliliters of 1x PBS to each tube and pipette up and down slowly to mix. Add an additional 6 milliliters of 1x PBS to the top of the tube and carefully mix again.

Ultracentrifuge the tubes at 100,000 x g and at 4 degrees Celsius to re-pellet the small vesicles. Decant the supernatant and tap the tubes dry before lysing the vesicles for protein analysis or resuspending the EVs for morphologic analysis.