Separation and Enrichment of Extracellular Vesicles in the Presence of Virions

Özet

Source: DeMarino, C., et al. Purification of High Yield Extracellular Vesicle Preparations Away from Virus. J. Vis. Exp. (2019)

The video demonstrates the separation of EVs from virions and other contaminants by density gradient centrifugation, followed by capture and concentration with hydrogel nanoparticles. Washed fractions are then ready for downstream identification of those enriched in EVs.

Protokol

1. Filtration and Precipitation of Extracellular Vesicles (EVs)

1. To prepare the culture supernatant from infected or transfected cells (i.e., cell lines and/or primary cells), culture approximately 10 mL of late-log cells for 5 days at 37 °C and 5% CO₂ in appropriate culture medium (i.e., Roswell Park Memorial Institute medium [RPMI] or Dulbecco’s modified Eagle’s medium [DMEM] with 10% fetal bovine serum [FBS]).
   NOTE: All culture medium reagents should be free of EVs, and can be either purchased (see Table of Materials) or prepared in-house by pre-ultracentrifugation of serum at 100,000 × g for 90 min. This protocol has been successful for several commonly-used cell lines including: CEM, Jurkat, 293T, U937 (uninfected lines), U1, J1.1, ACH2, HUT102, MT-2 (HIV-1 and HTLV-1 infected lines), multiple transfected cells, and primary myeloid and T-cells (both infected and uninfected); however, this protocol can be used for any cell type, including those that require specialized media or culture conditions. Density of cells may need to be optimized for different cell types. It is recommended that the highest density be used with minimal cell death after 5 days.
2. Centrifuge the culture at 3,000 × g for 5 min to pellet cells and discard the pellet.
3. Filter the culture supernatant using a sterile 0.22 µm filter and collect filtrate in a clean tube.
4. Add equal volume of PEG precipitation reagent (1:1 ratio) to filtered supernatant. Invert tube several times to ensure a homogenous mixture.
   NOTE: Do not vortex.
5. Incubate mixture at 4 °C overnight (O/N).
6. Centrifuge mixture at 1,500 × g for 30 min at room temperature (RT) to yield a heterogeneous EV pellet.
   NOTE: EV pellets should appear white or off-white in color.
7. Discard the EV-depleted culture supernatant.
8. Resuspend the EV pellet in 150–300 µL of 1× phosphate-buffered saline (PBS) without calcium and magnesium and keep on ice.

2. Construction of a Density Gradient

1. Mix iodixanol density gradient medium with 1× PBS to create 11 different 1 mL density fractions from 6 to 18% iodixanol in 1.2% increments in separate microcentrifuge tubes, as shown in Figure 1A.
2. Vortex each tube to mix.
3. Layer density fractions into a pre-cleaned and dry swinging bucket ultracentrifuge tube, starting with fraction #18 and ending with fraction #6 as indicated in Figure 1B.
   NOTE: All tubes should be sanitized using a 10% bleach spray followed by rinsing 3× with deionized water and a final wash of sterile deionized water prior to loading of the gradient fractions.
4. Add resuspended EV pellets (300 µL) to the top of the layered gradient in the ultracentrifuge tube.
5. Ultracentrifuge at 100,00 × g at 4 °C for 90 min.
6. Carefully remove the 1 mL fractions from the ultracentrifuge tube and transfer each fraction into new microcentrifuge tubes.

3. Enrichment of EV Fractions Using Nanoparticles

1. Create a 30% slurry of nanoparticles using equal volumes of NT80, NT82, and 1× PBS.
   NOTE: The mixture should be vortexed prior to use to ensure homogeneity.
2. Add 30 µL of the slurry to each microcentrifuge tube containing the density fractions and pipette/invert them several times to mix.
3. Rotate EV-enriching nanoparticle-containing density fraction microcentrifuge tubes overnight (O/N) at 4 °C at approximately 20 rpm.
4. Centrifuge density fraction microcentrifuge tubes at 20,000 × g for 5 min at RT.
5. Discard the liquid and wash the EV pellet twice with 1× PBS.
   NOTE: Nanoparticle pellets can be frozen at -20 °C or immediately used for various downstream assays (i.e., polymerase chain reaction (PCR), Western blot, mass spectrometry, and other assays).

Sonuçlar

Figure 1: Construction of an iodixanol density gradient. (A) Relative amounts of iodixanol and PBS utilized to create each density fraction (Fraction #). The Fraction # denotes the percentage of iodixanol ...

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
CEM CD4+ Cells	NIH AIDS Reagent Program	117	CEM
DPBS without Ca and Mg (1x)	Quality Biological	114-057-101
Exosome-Depleted FBS	Thermo Fisher Scientific	A2720801
Fetal Bovine Serum	Peak Serum	PS-FB3	Serum
HIV-1 infected U937 Cells	NIH AIDS Reagent Program	165	U1
Nalgene Syringe Filter 0.2 µm SFCA	Thermo Scientific	723-2520
Nanotrap (NT80)	Ceres Nanosciences	CN1030	Reactive Red 120 core
Nanotrap (NT82)	Ceres Nanosciences	CN2010	Cibacron Blue F3GA core
Optima XE-980 Ultracentrifuge	Beckman Coulter	A94471
OptiPrep Density Gradient Medium	Sigma-Aldrich	D1556-250mL	Iodixanol
SW 41 Ti Swinging-Bucket Rotor	Beckman Coulter	331362
Ultra-Clear Tube, 14 mm x 89 mm	Beckman Coulter	344059