Isolation and Characterization of Exosome-enriched Extracellular Vesicles from Dengue Virus type 2-infected Mammalian Cells

Summary

Here, we describe the isolation of exosome-enriched extracellular vesicles from Dengue virus type 2-infected cell culture supernatant, followed by characterization of vesicle integrity and size distribution, and evaluation for the absence of detectable infectious virus by plaque assay under defined experimental conditions.

Abstract

Exosome-enriched EVs are vesicles released from various cell types into extracellular fluids such as plasma, urine, saliva, cell culture media, and other body fluids. Their diameters range from 30 nm to 150 nm. They have been implicated in different cellular processes, including cellular communications and immune modulation during infection. Based on these effects, they have been used to study the pathogenesis of several viruses. It is challenging to isolate Exosome-enriched EVs from virus-infected cell supernatants using current purification techniques. This is largely due to the overlap in size between certain viruses and Exosome-enriched EVs. Here, we aim to purify Exosome-enriched EVs from Dengue type 2-infected cell culture supernatant. Exosome-enriched EVs were isolated using a commercial exosome isolation kit. For the isolated Exosome-enriched EVs in this protocol, no infectious virus was detected by plaque assay under the conditions tested. The purified exosome-enriched EVs were characterized using western blotting for tetraspanins, nanoparticle tracking analysis, and transmission electron microscopy for size confirmation. We demonstrated that our exosome-enriched EVs are intact and within the normal exosome diameter range of 30-150 nm. Overall, our protocol provides an inexpensive method for purifying exosome-enriched EVs from virus-infected cell culture supernatant, which can be used for downstream functional assays.

Introduction

Exosome-enriched EVs are nanosized vesicles released by every cell in the body. They are subtypes of extracellular vesicles (EVs) of endocytic origin. Exosome-enriched EVs were first discovered in reticulocyte culture media¹. In the past few decades, exosome-enriched EVs have been purified from several body fluids, including blood, urine, saliva, breast milk, seminal fluids, and amniotic fluids^2,3,4,5. They have a diameter that ranges from 30 nm to 150 nm⁶. Their release into the extracellular compartment occurs via fusion of multivesicular bodies (MVBs) with the cell plasma membrane. In the context of infections and immune response, exosome-enriched EVs have been known to elicit a specific response in mammalian cells. Such responses include the release of cytokines or alterations in susceptibility to infection^7,8,9.

The content of exosome-enriched EVs is influenced by several factors, most importantly, the type and state of the cell that produces them^10,11. Additionally, their content also determines the effects they have on naïve recipient cells. Several methods are currently used to isolate exosome-enriched EVs from biological fluids. Common methods include differential ultracentrifugation, precipitation, immunoaffinity capture, size exclusion chromatography, magnetic-based isolation, and microfluidic-based isolation.

Differential ultracentrifugation is considered one of the most utilized techniques^12,13,14. The ultracentrifuge purification method is expensive and requires significant technical proficiency. This method also results in a significant loss of EVs due to multiple ultracentrifugation steps. Finally, the resulting exosome-enriched EVs are not free of active viral particles because of their similar phenotypic properties. This has made purification of exosome-enriched EVs from virus-infected cell supernatant challenging. It is therefore important to develop an optimized protocol to effectively reduce dengue virus (DENV) levels to undetectable levels in the exosome-enriched EV solution, because viruses share similar sizes and densities with exosomes^15,16.

Compared with other commonly used techniques, this protocol offers several advantages, including reduced purification time, reliance on inexpensive and widely accessible laboratory equipment, and the ability to isolate exosome-enriched EVs with no detectable live viral contamination. Additionally, although methods such as density gradient centrifugation and magnetic-based isolation can offer advantages, including increased yield and higher purity, these approaches require an initial ultracentrifugation step. The aim of this article is to present a method of isolating exosome-enriched EVs from infected supernatants with no detectable infectious virus by plaque assay under the tested conditions.

Protocol

1. Preparation of exosome-free media

1. Attach a 0.1 µm filter to its receiver base inside the Class II biosafety cabinet (BSC).
2. Aseptically pour the heat-inactivated fetal bovine serum (FBS) on the filter and attach it to the vacuum pump inside the BSC.
3. Repeat step 1.2 with the flowthrough two more times.
4. Prepare complete media to contain 10% of the triple-filtered FBS, 1% penicillin/streptomycin, and 1% L-glutamine.
   NOTE: The triple filtration method is used to avoid the need for instruments such as an ultracentrifuge.

2. Dengue virus infection of mammalian cells

1. Grow Vero cells overnight in a T75 cm² culture flask inside a tissue culture incubator at 37 °C with 5% CO₂.
2. The following day, check to make sure the cell is between 70% and 80% confluent and infect the cells with 0.1 multiplicity of infection (MOI) of DENV-2 in 5 mL of complete media and incubate at 37 °C with 5% CO₂ for 2 h.
3. After 2 h, remove the supernatant and wash the flask 3x with sterile Phosphate Buffered Saline (PBS).
4. Add 50 mL of complete media into the flask and incubate at 37 °C with 5% CO₂ for 4 days (just before observing cytopathic effects).

3. Kit-based exosome purification

1. Transfer the cell culture media from the flask to a 50 mL conical tube and centrifuge at 200 × g for 15 min at room temperature.
2. Transfer the media to a new conical tube. Add 125 µL of EV precipitation reagent, followed by 600 µL of the resin. Vortex for 10 s and let it stand for 10 min inside the BSC at room temperature.
3. Vortex for another 10 s and centrifuge at approximately 450 × g for 2 min at room temperature. Transfer to the BSC, discard the supernatant, and apply 600 µL of resuspension buffer to the pellet.
4. Vortex for 10 s and incubate at room temperature for 15 min.
5. Vortex for another 10 s and centrifuge at approximately 25 × g for 2 min at room temperature.
6. Transfer the supernatant to a Filter Spin column and centrifuge at approximately 4,500 × g for 1 min at room temperature. Collect the flowthrough containing intact exosome-enriched EVs and store it at -80 °C until it is ready for use.
7. Confirm that the exosome-enriched EV is free of live virus by performing a plaque assay¹⁷.
8. Characterize the exosome-enriched EV using electron microscopy for size and nanoparticle tracking analysis for size distribution and concentration¹⁸.

4. Characterization of exosome-enriched EV by western blot

1. Determine the protein concentration of the exosome-enriched EV using assays such as BCA.
2. Mix equal volumes of exosome-enriched EV solution and RIPA buffer and add 4x LDS loading buffer (4:1).
3. Separate exosome-enriched EV proteins using a 4-12% Bis-Tris gel.
4. Transfer the proteins to a nitrocellulose membrane.
5. Block for 1 h using 5% non-fat milk dissolved in 1x TBS-Tween buffer at room temperature.
6. Dilute the primary antibodies using 5% non-fat dry milk to a 1:1,1000 dilution and probe the membrane for exosome proteins such as CD63 and CD9 at 4 °C overnight.
7. Remove the primary antibody and wash 5 x 5 min with 1x TBS-Tween buffer on a rocker.
8. Dilute the secondary antibody (anti Rabbit IgG HRP) with non-fat dry milk to a 1:5,000 dilution and incubate the membrane with this solution for 1 h at room temperature.
9. Remove the secondary antibody and wash 5 x 5 min with 1x TBS-Tween buffer on a rocker.
10. Develop the membrane using the ECL detection reagent and detect protein signals using the chemiluminescence imaging device.

5. Characterization of exosome-enriched EVs by transmission electron microscopy

1. Add 5 µL of exosome samples to the 200 mesh copper grids with formvar and carbon coating and allow to air-dry at room temperature.
2. Stain the grid with 1% aqueous uranyl acetate for 60 s and air-dry.
3. Capture the image using the TEM at 80 kV¹⁸.

Results

We analyzed the isolated exosome-enriched EV as described in the protocol using a plaque assay to determine if we could detect active DENV-2 particles in the mixture. As shown in Figure 1, no plaques were detected under the conditions tested when compared to the positive control. These results indicate that the infectious virus was below the detection threshold of the plaque assay. The plaque assay was done in biological triplicates and technical duplicates. ...

Discussion

Recent advances in infectious disease research have shown that exosome-enriched EVs released from virus-infected naïve cells play a critical role in modulating the host immune response^7,8,9. The exosome-enriched EVs can either enhance the immune defense by promoting an appropriate antiviral response or, conversely, facilitate viral immune evasion by suppressing host defense mechanisms.

To elucida...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti Rabbit IgG-HRP linked antibody	Cell Signaling	7074S	Secondary antibody
CD63	Cell Signaling	52090S	Primary antibody
CD9	Cell Signaling	13174S	Primary antibody
Cell culture media exosome purification kit	Norgen	60600	Exosome purification kit
Dengue virus 2	ATCC	VR-1584	Virus
DMEM	Fisher scientific	11-995-073	Culture media
EMEM	ATCC	30-2003	Culture media
FEI Talos F200X Transmission Electron Microscope	Thermofisher Scientific
Fetal Bovine Eerum	Neuromics	FBS002	Media Supplement
Nano particle tracking analyzer	Particle Metrix
Vero cells	ATCC	CCL-81	Mammalian cell line