Method for Isolation And Identification of Extracellular Vesicles
Extracellular vesicles (EVs) are a general term for various vesicles with a lipid bilayer membrane structure that are released under resting or stress conditions. The diameter of the vesicles ranges from tens of nanometers to several micrometers. Studies have found that extracellular bodies are involved in a variety of physiological processes in the body and are associated with a variety of pathological processes such as infectious diseases and inflammation, nervous system diseases and cancer. Therefore, extracellular vesicles can be used to monitor disease progression, therapeutic response, and the like. At the same time, because of their ability to deliver bioactive substances, it can also be used as a new nano drug carrier to deliver drugs. In view of the important biological functions and broad research and application prospects of EV, EV has received extensive attention as an emerging field in recent years. However, because EV is very small in size, it is challenging to separate, observe and identify it. Therefore, finding efficient EV separation and identification methods is the prerequisite for research.
Separation method
EV is small in size, low in density, difficult to separate, and different EV physical and chemical properties obtained by different separation methods, which leads to a large amount of uncontrollability and difficulty in repeating EV research, which is the most important factor restricting EV research and application transformation. Many separation techniques have been developed using the physical and biochemical properties of EV, such as ultracentrifugation, density gradient centrifugation, immunoadsorption, precipitation, microfluidic-based separation techniques, and the like. However, in all known methods, the methods which can extracte quickly, simply and efficiently EV, and ensure its morphology, purity, yield and biological activity conforms to the requirements of subsequent experiments is not exsit. At present, the commercial precipitation kit extracts exosomes quickly and has high yield, but the price is relatively expensive, the reagent residues are more, and there is certain cytotoxicity, which has a great influence on the subsequent functional tests. Only by integrating the existing extraction methods can the impurities in the sample be effectively reduced. At present, the commonly used method for extracting exosomes is mainly for the study of bioactive substances in exosomes. Once applied to clinical treatment, methods for effectively improving the purity and yield of exosomes need to be solved urgently.
Figure 1. Working principle of common methods to isolate extracellular vesicles.
| Separation method | Working principle | Advantage | Disadvantage |
| Ultracentrifugation | Separation is based on the size of the EV, with large precipitating at the bottom of the tube and small requiring more centrifugal force to precipitate. Soluble components are not affected by centrifugation, but non-EV particles such as lipoproteins and protein aggregates may precipitate together | Separation of exosomes with high purity is the current gold standard for separation of exosomes, most commonly used | The instrument is expensive, technically difficult, time-consuming and labor-intensive, and the output is low. Repeated centrifugation may cause damage to the EV and affect the quality. |
| Density gradient
Centrifugation |
Separation based on EV density, EV can be separated from particles of different densities, EV will move to its equilibrium density, and high-density soluble components will precipitate at the bottom of the tube | Low density EVs can be separated from other vesicles, particles and contaminants, resulting in higher EV purity | The steps are cumbersome, time consuming and extremely sensitive to centrifugation time |
| Molecular volume exclusion chromatography | Separation was performed using a porous gel matrix based on the size of the EV molecule. The soluble component and particles having a particle size smaller than the critical value enter the porous matrix, while the EV and particles larger than the critical value do not enter the porous matrix and elute rapidly. This method can accurately separate large and small molecules | The EV isolated by this method has high purity and is uniform in size under electron microscope, and is not affected by the shear force that may change the structure of the vesicle. | Need special equipment, time-consuming and labor-intensive, less harvest, not widely used |
| Ultrafiltration | EV is larger than normal protein, and the sample is selectively separated by ultrafiltration membranes with different molecular weights. The soluble protein and particles smaller than the critical value (about 105 kDa) are pushed to the filter membrane, and then the EV is collected at the filter membrane. | Simple and efficient, it separates EV from small particles and soluble molecules without affecting their biological activity | EV may be attached to the filter membrane and lost. When ultrafiltration passes through the membrane, the pressure may cause EV deformation or destruction, which may be contaminated by protein. |
| Immunoadsorption | Based on the EV surface-specific label, the EV can be adsorbed and separated by incubation with the EV coated with the corresponding antibody. | Based on the EV surface-specific label, the EV can be adsorbed and separated by incubation with the EV coated with the corresponding antibody.Simple operation, high specificity, no influence on EV morphology, purity, and EV obtained directly for analysis or for DNA or total RNA isolation | Inefficient, exosome biological activity is susceptible to pH and salt concentration, which is not conducive to downstream experiments. Antibodies are expensive and non-recyclable, and are not suitable for obtaining EV from a large number of samples. |
| Precipitation | The polymer-based precipitant is co-precipitated with hydrophobic proteins and lipid molecules to separate the EV, and non-EV particles and soluble proteins can also be aggregated and precipitated. One of the most common polymers is polyethylene glycol. | Easy to operate, low technical difficulty, short time | Low purity and recovery, high amount of heteroproteins (false positives), uneven particle size, difficult to remove polymer, mechanical force or Tween -20 ° C and other chemical additives will destroy exosomes |
Identification MethodIn addition to the above methods, some scholars have tried to use ion resonance biosensors, nanolipid probe systems, microfluidic systems, cholesterol-based heat-assisted acoustic fluid separation vesicles, spin ultrafiltration, and immunomodification. Technologies such as superparamagnetic nanoparticles provide new avenues for rapid, efficient and high-purity EV separation and elution, which are beneficial for the application of exosomes. The EV produced by this method has good stability and is easy to store.. A common method of EV preservation is to resuspend it in a sterile phosphate buffered saline (PBS) solution, which can be stored for one year at -80 °C without changing its morphology and biological properties. Stored at -20 °C for 6 months. Recent research has shown that a low pH acidic environment is conducive to stable storage of EVs and can increase EV production.
At present, the identification methods for EV (mainly exosomes) include morphology, particle size, surface markers, etc.as follows:
1. Antibody-Based Identification Method
Since EV is produced in the cell membrane pathway, antibody-targeting markers associated with this pathway can be identified. These include the four transmembrane protein superfamilies (CD9, CD63 and CD81), AIP1/Alix, TSG101 and CD326/EPCAM. Methods for protein identification can be performed using Western blotting. This method can be used in conjunction with some of the techniques described below to determine the population of EVs.
2. Transmission Electron Microscope
Transmission electron microscopy can be used to observe the surface characteristics of the EV. The purified EV was placed in a suspension, placed on a microscope sample, and negatively stained with uranium acetate and cellulose. The transmission electron microscope can clearly show the morphology of the EV. Under the microscope, EV presents a bilayer-enveloped vesicle structure, often described as a “cup shape,” but this may also be an artifact caused by drying when processing a sample. Therefore, this method should not be used as a clear feature of EVs, nor as an identification of EV sources. In the preparation of electron microscopy, the above antigens can be single or double stained, and then used Gold nanoparticles of different sizes with antibody specificity were used for secondary staining. In electron microscopic images, these nanoparticles can clearly distinguish between different sizes of EV. In addition, standard tissue electron microscopy techniques can be used for identification, and a more accurate assessment of EV presence can be obtained by combining antibody and tissue sections.
3. Identification Method Based on Particle Size
One of the most common methods for identifying EVs based on particle size is NanoSight Nanoparticle Tracking Analysis (NTA). This technology is to install a high-definition camera on an optical microscope. Using the properties of light scattering and Brownian motion, specific exosomes and microvesicles in the range of 50 ~ 1000 nm are directly imaged and observed one by one. The high resolution particle size distribution data and concentration information can be used for semi-quantitative detection of exosomes. Unlike TEM, there is no need for pre-treatment such as drying, fixing, and freezing. The NTA can be tested in situ and closer to its original state, providing structural and functional protection for EV particles, ensuring the authenticity of measurement data and effectiveness.
4. Flow Cytometry
EV Because the particles are too small, lower than the threshold of conventional flow cytometry analysis, it is not possible to accurately distinguish between particles and noise. Exosome fluorescence labeling can be recognized by flow cytometry, but the number of particles cannot be quantified because the “swarming effect” instrument itself cannot accurately separate particles from noise. An alternative approach is to bind the EV directly or indirectly with an EV-specific antibody using latex microspheres. This antibody-bound latex microsphere can be made by itself or commercially. The bound EV can then be labeled with other fluorescently bound specific antibodies. Since it is not clear how many particles are bound to each latex microsphere, the number of particles is still not directly available, but this method is used to analyze the surface antigen of EV.
5. Fluorescence and Confocal Microscopy
The EV can be labeled with a lipophilic film-bonded dye (such as PKH67, DiD, etc.), or the EV can be marked with a thiol on its surface. This technique does not really visualize every EV, but it can be used for research on whether labeled EVs can be taken up by cells.
6. Other Methods
Other methods for detecting a single EV include atomic force microscopy, field emission scanning electron microscopy, Raman spectroscopy, micronuclear magnetic resonance, small-angle X-ray scattering, anomalous SAXS, and resistive pulse sensing.