Article Author: Alba Calzas Delgado (I+D+i Immunostep Department)
Exosomes have recently emerged as a new source of potential non-invasive biomarkers of several diseases, since they can be easily obtained from body fluids such as urine, blood, saliva, or breastmilk and their composition may be directly dependent on the physiological and/or pathological state of the patient. In this sense, cancer and neurodegenerative diseases are having the greatest impact on the analysis of these vesicles. For this reason, in this article we aimed to collect and compare the performance of each of the most commonly used exosome isolation methods in order to provide useful guidelines on the most appropriate method depending on the type of sample and starting volume.
The isolation and characterization of exosomes from biological fluids can provide valuable information for early detection, disease monitoring and treatment development.
Research on exosomes as a potential source of biomarkers of human disease has increased rapidly in recent years and consequently there are a large number of techniques for the isolation and characterization of this type of extracellular vesicles. However, many of the current techniques are poorly standardized. Furthermore, the use of exosomes in diagnostic tests or clinical research requires a sensitive, reproducible, and high-throughput method for the detection, characterization, and quantification of exosome samples.
Exosomes are small (~40‐150 nm) extracellular vesicles (EVs) released from all cell types and found in body fluids and cell culture supernatants. These vesicles participate in intercellular communication without the need of direct cell-to-cell contact, since their contents travel long distances through the bloodstream, lymph or other fluids.
Exosomes differ from the rest of EVs in their size , their endocytic origin and their content; therefore, they are nanovesicles consisting of a lipid bilayer, whose biogenesis is based on fusion of the multivesicular body (a specialized endosome) with the plasma membrane, and whose molecular composition is also different from the rest of EVs.
The biological characterization of exosomes requires in most cases the isolation of intact exosomes. In this regard, a wide variety of methods have been developed for the isolation of exosomes from biological fluids, including ultracentrifugation, size exclusion chromatography, immunological separation and precipitation by polyethylene glycol. Each of these methods has its advantages and disadvantages: time needed to perform the method, the need for specialized equipment, sample starting volume, purity and recovery are key parameters to consider in this regard.
Differential centrifugation is a process widely used in microbiology and cytology, which is used to separate some elements of a cell for further analysis of specific parts of the cell. To do this, the sample is first filtered or clarified to remove debris and cells, and then subjected to repeated centrifugations, progressively increasing the centrifugal force. Finally, the extracellular vesicles fraction to be analysed is recovered.
Size exclusion chromatography is a technique for the isolation of macromolecules in which a porous gel allows the separation of different elements (solutes) according to their molecular sizes. The material used is composed of small particles of silica or polymers, which have pores where the molecules are trapped, so that the larger ones are retained and elute quickly, while the smaller sizes can penetrate them and are retained for a longer period of time.
The main advantage of this method is that exosomes are isolated in a single step, the protocol takes little time and allows the removal of contaminating proteins efficiently, as well as the separation of exosome subtypes. As a result of this process, extracellular vesicles isolated by this method are obtained with high purity.
Some of the disadvantages associated with size exclusion chromatography are that samples are collected in several fractions, and these have to be characterized to ensure the presence of extracellular vesicles and proteins. In addition, aseptic working conditions are necessary to avoid microbial contamination and a large sample loading volume can lead to inefficient separation, so small sample volumes are necessary for optimal results. (Immunostep Exosome Isolation Columns)
A precipitate is a solid produced by a solution as a result of a chemical or biochemical reaction, this process is called precipitation. When an insoluble substance is formed in a solution, or the solution is supersaturated by some compound, precipitation is formed: if the precipitate is denser than the rest of the solution it falls, if it is less dense, it floats, and if it has equal density, it remains in suspension. (Immunostep Exosome Precipitation Solutions)
The main advantage of this method is the high yield of the procedure, as well as the ability to precipitate practically all the extracellular vesicles in the sample. The method is fast and simple. On the other hand, precipitation combined with protein solvents can improve purity and ensure contaminant-free isolates for therapeutic applications.
Disadvantages of the precipitation method include the need to remove the polyethylene glycol or precipitating salt to prevent it from interfering with downstream applications, as well as the need for additional purification steps.
Immunoaffinity-bead based EV isolation techniques employ antibodies to target biomarkers present in the surface of the exosome, to provide higher specificity and purity in the isolation than other traditional methods.
Potential subpopulations of exosomes can be captured by targeting typical exosome markers using beads. This protocol allows to further improve the purity of the exosome preparation and can be applied directly on the sample, avoiding the pre-enrichment step.
The direct immunoaffinity-bead based isolation is user friendly and produces highly purified exosome preparations with minimal loss and is a versatile tools for exosome isolation and downstream analysis (Immunostep Exosome Capture Beads).
The initial sample volume has a considerable effect on the isolation and detection techniques used. In this sense, ultracentrifugation remains by far the most widely used primary isolation method for all applications, while the Western Blot (WB), the Nanoparticle Tracking Analysis (NTA) and electron microscopy, in this order, are the preferred techniques for the analysis and characterization of exosomes.
However, none of these detection techniques meets the premise of being reproducible and offering high performance potential, for diagnosis or clinical research application.
Flow cytometry is a technique well adapted to the reproducible analysis of clinical samples, allowing the analysis of different physical and chemical characteristics of cells and particles in suspension.
In this scenario, most researchers usually use more than one method for the characterization of exosomes in their experiments.
Specifically, the quantification and size analysis of the vesicles is done by NTA and dynamic light scattering (DLS), although these techniques are limited to the definition of the size range and concentration of the exosomes without contributing much to phenotype information.
Likewise, to determine the protein composition, most researchers perform WB technique, despite being a laborious and non‐quantitative method that sometimes yields poor results, due to the quality of the antibodies used and/or to low exosome abundance in the sample.
In order to overcome some of the limitation we have describe in this article, at Immunostep we developed a specific, sensitive and easily scalable method and kit for exosome detection and characterization from supernatants of cell cultures and biological fluids by flow cytometry. This method is based on the use of magnetic beads coated with antibodies against tetraspanin CD63, a common marker of exosomes, which allows exosome detection in conventional cytometers. (ExoStepTM Kit)
Traditionally, exosomes have been isolated by differential centrifugation from culture medium whereby larger contaminants are first excluded by pelleting out through increasing speeds of ultracentrifugation before exosomes, small extracellular vesicles and even protein aggregates are pelleted at very high speeds. These preparations therefore represent an enrichment rather than a purification. In this sense, we build technology to improve the performance of these traditional methods to isolate and characterize exosomes derived from biological fluids.
Although we can isolate exosomes with any of the three methods described above, there are more appropriate methods depending on the sample and volume from which we start, as well as its subsequent application. Our experience and knowledge in dealing with these types of exosome isolation methods throughout our career has shown us the following:
► The exosome precipitation solution is framed within the polyethylene glycol-based precipitation methods, and its main advantages include its simplicity, speed, reproducibility. After performing several tests, with different types of samples and different starting volumes, we can affirm that for a plasma or urine sample with a volume between 1-20 mL, if what we want to perform is a protein analysis or to know the concentration of exosomes contained in the sample, this method is the most effective. Likewise, if we have a smaller sample volume and it comes from a sample of CSF, milk, etc., and what we want to perform is a protein analysis, this technique is also advisable.
► Ultracentrifugation is still by far the most widely used primary method, although it is a time-consuming method that yields aggregated proteins and nucleic acid can sediment, we have found it to be a suitable technique for processing any type of sample with a volume greater than 20 mL and for cell cultures with volumes between 1-20 mL. We have also found that it is not a very suitable technique for small sample volumes.
► Size exclusion chromatography (SEC) has been described as the most efficient method to isolate extracellular vesicles in a single step, with good recovery and almost complete removal of contaminants such as proteins and lipoproteins. It is a very efficient assay if we are looking to perform nucleic acid analysis or in vitro assays from a plasma and/or urine sample volumes of less than 1 mL up to a maximum volume of 20 mL. Finally, if we start from a sample volume greater than 20 mL, we could also combine techniques: perform a differential ultracentrifugation and then the method that best suits us depending on the purpose of its application.
► All the methods can be combined and, it is really interesting to combine bead-based assays to detect exosomes after isolate with other isolation techniques in order to improve the results and purity of the isolation.
If you are interested in obtaining help to find the most efficient exosome isolation method for your research or you would like to receive further information about our products please contact us.