Exosomes: Isolation and Investigation
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What are exosomes?
Exosomes are small vesicles of 30 to 150 nm. They are found in extracellular medium and bodily fluids such as blood, saliva, urine and so on. Their purpose is to transport lipids, proteins, nucleic acids and other macromolecules to recipient cells. Initially they were considered to be “waste products”, however, it is now well-established that they play an essential role in inter-cell communication.
They are receiving an increasing amount of attention due to their involvement in the development and progression of several diseases, including neurodegenerative diseases, liver disease, and cancer.
To investigate their function there is a need to optimize and standardize the methods employed to isolate exosomes. However, before exploring the different methods, it is worth explaining what makes exosomes so interesting…
Why are exosomes important?
There are three main reasons why exosomes are important:
- They participate in inter-cell communication.
- They have been attributed roles in the spread of proteins, lipids, mRNA, miRNA and DNA that have been linked to normal and pathological processes.
- They have been proposed as potential vectors for drug delivery. They would encapsulate their therapeutic “cargo” with a cell membrane rather than a synthetic polymer which would be better tolerated by the host.
Exosomes and cancer
There is mounting evidence to suggest that exosomes are involved in cancer progression. They participate in the inflammatory response, angiogenesis, lymphogenesis, cell migration, cell proliferation, immune suppression, invasion, epithelial-to-mesenchymal transition and even metastasis. Their implication in cancer progression makes them well-suited tools for tracing and tracking cancer. For example, the increasing quantity of exosomes in body fluids can be an indication of the disease. Research seem to suggest that there is a relationship between the number of exosomes secreted and whether the “state” of the cell – whether it is “normal” or cancerous. For example, higher-grade cancer cells secrete a higher number of exosomes than low-grade cancerous cells. The contents of exosomes derived from cancerous cells vs “normal” cells is also different. These areas still require further exploration to gain a deeper understanding of the relationship between exosomes and cancer.
Exosome isolation and purification methods
Several methods have been developed for optimal exosome separation.
The main advantages and disadvantages of the different methods available are presented in the table below.
|Exosome Isolation Method||Mechanism||Advantage||Disadvantage||Examples|
|Differential Centrifugation||The different particles present in the biofluid are separated by differential centrifugal force.|
It is the most common approach used to isolate extracellular vesicles (EVs).
It is cost-effective and very flexible in the scale of production.
It separates microvesicles, apoptotic bodies and exosomes from biological fluids.
It requires specialized instrumentation such as ultracentrifugation and rotors.
Isolation efficiency varies according the instrument used.
The multi-step process is time consuming.
Biological fluid needs dilution prior to centrifugation because of its
|Density Gradient Centrifugation||It combines ultracentrifugation and density gradients to separate exosomes from other particles of different densities.||Further purifies the extracellular vesicles from co-precipitation such as protein aggregates, apoptotic bodies or nucleosome fragments.|
Needs extra washing steps.
Doesn’t separate certain lipoproteins and virus vesicles from extracellular vesicles since they share similar densities.
|Ultrafiltration||It uses membranes of different pore sizes to separate EVs.|
Enriches EVs from small biofluid volumes.
The procedure is easy and quick.
May result in exosome retention because of membrane-protein binding or saturation of the membrane pores.
The filtration force may result in vesicle rupture.
|Polymer-based Precipitation||It uses volume-excluding polymers to wrap and aggregate EVs.|
Doesn’t require specialized instruments.
The process of isolation is easy to handle.
It is high-throughput and efficient for EV isolation.
The final pellet may contain lipoproteins.
The presence of polymers may affect subsequent analysis.
|miRCURY; ExoQuick and Total Exosome Isolation Reagent (Invitrogen)|
|Size Exclusion Chromatography (SEC)||It is based on a column of beads with different pore sizes. The EVs are separated and collected based on their different hydrodynamic radii passing through the column at different rates.|
The isolation of EVs is done in a single step.
The integrity and biological activity of the particles is altered slightly.
Doesn’t concentrate EVs.
Optimization and re-equilibration are needed before running which limits size exclusion chromatography (SEC) efficiency.
|Izon qEV SEC|
|Immunoaffinity Capture||Antibodies recognize the antigen expressed on the surface of EVs.||It can be used to isolate total number of EVs or a specific group of EVs expressing a unique antigen.|
It is not suited for a large sample volume.
There is heterogeneity in antigen expression.
The antibody needs to be available.
|MACSPlex Exosome Kit; Exo-Flow|
|Microfluidic Device||Uses distinct or combined technologies such as immune affinity, SEC and filtration to study the EVs present in the biofluid at microscale.|
It works well for small sample volumes.
High efficiency, high Sensitivity and low cost sample analysis.
The high cost of the device is a major disadvantage.
Further developments and optimization is needed for it to become a clinical tool.
|iMER, nPLEX; NanoDLD|
All in all, current definition of exosome is based on their size and their implication in inter-cell communication. It has been suggested that they might be signal amplifiers for cancer and potential tools to detect and treat several diseases. However, to better understand these extracellular vesicles, researchers must first “master” exosome isolation and purification.
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