Extracellular vesicles, also known as extracellular vesicles (EVs), are nanoscale vesicles actively secreted by all cells. Living cells release different types of extracellular vesicles into the extracellular environment for intercellular communication, and extracellular vesicles are increasingly considered promising biomarkers for liquid biopsy. Based on the diameter and size of similar vesicles, extracellular vesicles can be divided into three categories, with diameters ranging from 50-150nm, Microvesicles, exosomes, and microparticles with a diameter of 100-1000nm, as well as apoptotic bodies with a diameter of 100-5000nm. Currently, it is mainly believed that the process of exosomes production is the invagination of the cell membrane to form endosomes, which then form polyvesicles. The fusion of polyvesicles with the plasma membrane leads to the release of vesicles from the lumen to the extracellular vesicles, producing a subtype of EV called exosomes
Extraction and purification methods of extracellular vesicles
2.1 Density based separation method
2.1.1 Overspeed centrifugation method
The ultracentrifugation method is the most commonly used method for extracting extracellular vesicles. Firstly, a lower centrifugal force of 300g is applied to remove cells from the cell culture medium; Then, a large centrifugal force (10000-20000g) was applied to the supernatant to remove large cell fragments and broken Organelle; Finally, high-speed centrifugation (100000-150000 g) was performed again to collect extracellular vesicles from the supernatant. All centrifugations were performed at 4 ℃. The extracellular vesicles obtained by the high-speed centrifugation method were not contaminated by separation reagents, and the number of separations was large, resulting in a small sample size. Although the high-speed centrifugation method is the most widely used "gold standard" for extracting extracellular vesicles, it still has many drawbacks, If the required ultra high speed centrifuge instrument is expensive, with a large sample size, long time consumption, and protein contamination still exists when observing exosomes under electron microscopy
2.1.2 Sucrose density gradient centrifugation method
At present, it has been found that exosomes float in a sucrose gradient of 1.15-1.19g/mL density. Therefore, based on this characteristic, the sample can be ultracentrifuged together with a sucrose gradient solution, and the exosomes can be distinguished by settling to different density regions. The sucrose density gradient centrifugation method requires pre preparation of continuous gradient concentration sucrose solution, laying the sucrose solution at the bottom of the centrifuge tube, and then placing the sample on the upper part, 100000g ultracentrifugation at 4 ℃. The purity of extracellular vesicles obtained by sucrose density gradient centrifugation is relatively high, but the preliminary preparation is complex, time-consuming, and cannot completely separate the extracellular vesicles from proteins. Some researchers at the ISEV meeting in October 2013 stated that the biological function of cell vesicles is lost when using sucrose density gradient centrifugation to separate vesicles
2.2 Precipitation method
2.2.1 Polyethylene glycol (PEG)
PEG is a water-soluble non Ionic compound with strong hydrophilicity, which can combine with hydrophobic lipid bilayer to change the solubility of exosomes and precipitate exosomes. RIDER and other studies found that the level of PEG will affect the yield of exosomes, and the total protein and RNA obtained from exosomes are sufficient in quantity and quality for Proteomics and sequencing analysis. The precipitation method is simple, does not need special equipment, and is more economical, The production of exosomes is high, but some hydrophobic substances that are not exosomes will precipitate, resulting in insufficient purity of exosomes
2.2.2 Reagent Kit Method
Recently, reagent kits based on polymer co precipitation have been developed, such as ExoQuick, TEI, etc., which can be used to extract extracellular vesicles from various body fluids. The polymer precipitant ExoQuick is co incubated with the sample at 4 ℃ for 30 minutes, and then centrifuged at room temperature of 1500g for 30 minutes to obtain extracellular vesicle precipitation. Compared with ultracentrifugation, the reagent kit method is simpler, shorter in time, and can achieve higher extracellular vesicle production. The reagent kit method contains more impurities in the extracellular vesicle precipitation, Samples from different sources require different reagent kits for extraction, and the cost of the kits is relatively high
2.3 Size based separation method
2.3.1 SEC
The exosomes are mainly separated and purified according to the size of the exosomes. Macromolecules in the sample cannot enter the gel pore but are quickly washed out by the mobile phase. Substances smaller than the pore size can enter the porous material and need to be washed out for a long time, so the exosomes can be separated by different elution times. BING and others have proved that agarose gel can purify exosomes from platelet free supernatant. By this method, Exosomes can be easily separated from proteins and High-density lipoprotein. HONG and others can effectively separate exosomes by adapting and using the mini SEC method. Unlike the long and complex ultracentrifugation method, it can complete exosomes separation within 30 minutes. The exosomes separated by SEC have high purity, and the separation of structurally complete and functionally active vesicles is an important advantage based on the micro SEC separation, but the number is small and requires special equipment, Therefore, it is not widely used
2.3.2 Ultrafiltration method
Ultrafiltration method uses a filter membrane with corresponding pore size based on the size of the exosomes to filter small and medium-sized molecules from the sample to the other side of the membrane, while retaining large molecules on the membrane to achieve separation. Ultrafiltration method is simple, time-saving, and low-cost. LIU and others have improved the simple ultrafiltration method by connecting membranes with different pore sizes (200, 100, 80, 50, 30nm) in series, achieving rapid separation of exosomes of different sizes, And the capture efficiency is significantly higher than that of the ultracentrifugation method. However, the filter is easily blocked by vesicles and other macromolecular substances, which can easily lead to excessive membrane pressure and breakage
2.4 Separation method based on surface component affinity
2.4.1 Protein
The surface of exosomes contains rich proteins, so the affinity based on their surface components is particularly suitable for separating exosomes. CD63 is one of the most abundant proteins found in exosomes, and therefore, anti CD63 immunoadsorbent exosomes are commonly used. ZHAO and others use anti CD63 coated magnetic beads to continuously mix with blood samples, capture the exosomes onto the magnetic beads, and rinse with buffer for 5 minutes, Then three different fluorescent dye labeled antibodies [anti CD24, anti epithelial Cell adhesion molecule (anti EpCAM), anti carbohydrate antigen-125 (anti CA-125)] were introduced, and the expression levels of different tumor markers in ovarian cancer could be quantified by observing different fluorescent intensities
2.4.2 Membrane phospholipids
Although most surface composition based affinity methods are based on proteins on the surface of exosomes, lipid bilayers are also a good detection target. XU et al. utilized the expression of phosphatidylserine (PS) on the exosomes membrane to bind well to the PS binding receptor Tim4, and used Tim4 immobilized magnetic beads to react with the sample for exosomes capture. It was observed that the eluted exosomes remained intact, Compared with commercial exosomes extraction kits, CHEN et al. exhibit higher capture rates. CHEN et al. utilized the characteristic of exosomes exposing negatively charged PS onto the membrane, using magnetic beads of ion exchange resins with positively charged groups to react with plasma samples. The exosomes in plasma can bind to the magnetic beads, and the exosomes separated by this method have higher recovery rates and fewer impurity proteins compared to ultracentrifugation
2.5 ACE separation method
The Dielectrophoresis (DEP) separation force generated by the ACE microarray is generated by applying an AC electric field. Nanoparticles and other nano solid substances are attracted to the DEP high field area around the edge of the circular microelectrode, and cells and large solid substances are attracted to the DEP low field area. The ACE devices such as IBS EN require 30-50 μ L plasma samples can concentrate extracellular vesicles into the high field area around the microelectrode within 15 minutes. The ACE equipment process is significantly faster than the currently used methods, and this device simplifies the ability to extract and recover extracellular vesicles, significantly reducing processing steps and consumption time. CHEN and others have constructed a DEP chip with cross electrodes, which can separate extracellular vesicles from plasma samples within 30 minutes. After testing, it has been proven that, DEP chips have high capture and recovery rates, require shorter time, and do not require bulky and expensive instruments
2.6 Microfluidics chip method
The Microfluidics chip method is a newly developed method for rapid and efficient separation of exosomes from samples. WOO and others used the experimental disk integrated with two nanofilters (Exodic) to achieve full automatic enrichment of exosomes of 20-600nm within 30 minutes. The quantitative detection using nanoparticle tracking analysis confirmed that the recovery rate of exosomes from cell culture supernatant was more than 95%. Compared with ultracentrifugation, Exodic provided a mRNA level 100 times higher, which was more time-saving, Less sample size is required. FANG et al. have developed a microfluidic chip, passing magnetic beads coated with anti CD63 and plasma samples into the chip, capturing exosomes in the first chamber, passing in the first antibody to combine with the magnetic bead exosomes mixture, and then passing in the fluorescent labeled second antibody to form a magnetic bead exosomes first antibody second antibody mixture to gather in the second chamber. The Microfluidics chip method is simple in operation and has high capture rate, Especially suitable for biological research. As a promising biomarker for cancer diagnosis, exosomes have received attention in liquid biopsy of cancer. The biological and clinical value of exosomes highlight the importance and necessity of developing effective extraction and separation techniques for exosomes. It is believed that with the continuous progress and innovation of technology, exosomes extraction will become more convenient and economical, with higher purity and better integrity
After extraction, further testing is often required to determine whether the extracted exosomes are exosomes. There are three methods: 1. Scanning electron microscopy observation; 2. NTA instrument particle size detection; 3. WB detection. As shown in the figure, there are often many biomarkers on the extracellular vesicles, so corresponding antibodies can be selected for WB detection. According to 22 articles related to exosomes, the top 4 detection indicators are CD63 (13/22), Tsg101 (8/22), CD9, and CD81, ranking third (6/22); The four indicators that were detected more frequently were Alix (4/22), HSP70 (3/22), flotillin (3/22), and Synthenin (2/22); In addition, some indicators have only appeared in one literature, such as HSP90, LAMP2B, LMP1, ADAM10, niastrin, AChE, AQP2, RPL5, a-1AT. Selecting at least two indicators for qualitative detection of extracellular vesicles can meet the needs of article publication, such as detecting CD63 and Tsg101.