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 ....

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 V....

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