Q&A of Extracellular Vesicle Research

Some articles indeed use serum-free culture medium to culture cells and then collect the supernatant for EV isolation. The method involves culturing cells with EV-containing serum until reaching a certain density, removing the culture medium, washing the cells with 1×PBS several times, and then switching to serum-free culture medium. However, many high-impact articles currently directly use serum-free culture medium for cell culture in EV research.

It is recommended to use culture medium without EV-depleted serum for cell culture. If resources are limited, cells can be initially cultured with serum-containing medium. Once the cells reach an appropriate density, around 70%-80%, the culture medium can be removed. The cells can be washed with pre-warmed PBS at 37°C for 3-5 times, followed by switching to serum-free culture medium. The culture supernatant can then be collected for EV isolation.

The choice of isolation and purification method depends on considering downstream experiments comprehensively. For serum or plasma samples, if downstream studies involve proteomic experiments, it is necessary to remove highly abundant lipoproteins from EVs. Size exclusion chromatography (SEC) is recommended in such cases. For nucleic acid experiments, there are multiple options available, but the relationship between sample quantity and EV yield needs to be considered. Nucleic acid experiments generally require a larger amount of EVs compared to proteomic experiments. If the sample quantity is sufficient, ultracentrifugation or SEC can be chosen. If the sample volume is less than 1 ml, precipitation with polymers is recommended. Each method has its advantages and disadvantages. Here are some examples for comparison:

• Ultracentrifugation: This method is classic but requires special equipment, involves complex procedures, has high technical difficulty, poor repeatability, and is time-consuming. It provides high EV purity but has limited effectiveness in removing lipoproteins.
• Polymer precipitation: This method is simple to operate, has low technical difficulty, is time-saving, yields lower EV purity but higher yield.
• Size exclusion chromatography (SEC): It effectively removes high-density lipoproteins (HDL) and low-density lipoproteins (LDL), resulting in high EV purity and good repeatability. However, the yield is relatively lower, and it offers easy standardization of operations.

According to the statistics from EV-related literature, the top four commonly detected markers are CD63, TSG101, CD9, and CD81, which are tied for the third position. The next frequently detected markers are Alix, HSP70, flotillin, and Syntenin. Additionally, there are some markers that appear in only a few studies, such as HSP90, LAMP2B, LMP1, ADAM10, AChE, AQP2, RPL5, and α-1AT.

For qualitative detection of EVs, it is recommended to choose three positive markers, CD9, CD81, and CD63, and one negative marker, calnexin.

Currently, most literature does not include the detection of reference genes. In some individual studies, Actin, GAPDH, and Tubulin have been used as reference genes for detection. However, based on the literature survey results, the expression abundance of Actin, GAPDH, and Tubulin in EVs is relatively low compared to normal cell samples. Therefore, when performing Western blot, it may not detect any bands or the bands may be very weak. Therefore, it is more common to use equal protein quantification to calibrate the differences between samples.

It is better to use plasma samples for EV research. Serum is the liquid collected after blood coagulation, which lacks fibrinogen, clotting factors, and contains many clotting by-products. Studies have found that during the clotting process, platelets secrete a large number of EVs, and nearly 50% of the EVs in serum are derived from this additional secretion. Additionally, when serum coagulates, a fibrin mesh is formed, which entraps EVs originally present in the blood. This positive and negative effect results in a lower association between EVs in serum and conventional diseases compared to plasma. Furthermore, Plasma = Blood - Blood Cells; Serum = Plasma - Fibrinogen - Clotting Factors. Therefore, in specific situations, such as studying platelet-related diseases, serum may be more suitable.

Research on EVs mainly includes three directions:

Disease mechanisms/drug resistance research: This focuses on studying EVs as shuttles between cells, mediating intercellular communication and playing a role in immunity. It falls within the scope of microenvironment research.

Biomarker research: This involves studying the relationship between the types, numbers, sizes, and contents of EVs and diseases. It quantifies the proteins and nucleic acids in EVs to serve as biomarkers for predicting the occurrence and progression of diseases.

Drug research: In the study of targeted drugs, EVs can serve as carriers to deliver drugs encapsulated within them to the target area.

The volume of cell culture supernatant used depends on the cell line type and the condition of the cells. Generally, stem cells, such as mesenchymal stem cells (MSCs), have a relatively strong ability to secrete EVs. For ultracentrifugation-based EV isolation, it is recommended to start with a cell culture supernatant volume of 50 ml or more for initial exploratory conditions, while for kit-based methods, a starting volume of 10 ml or more is recommended. Based on the results of exploratory experiments and the desired EV dosage for downstream experiments, the corresponding volume of supernatant can be used.

The sample volume and method selection for isolating EVs from serum and plasma depend on the desired EV dosage for downstream experiments. Generally, due to the relatively high content of EVs in serum and plasma samples, a starting sample volume of >200 μl is recommended for completing a proteomics experiment using a kit-based method. For size exclusion chromatography, a starting sample volume of >500 μl is recommended, while for ultracentrifugation, a starting sample volume of >2 ml is recommended. Other experiments may require preliminary explorations, which also depend on the downstream detection techniques. In general, the sample volume for gene chips is usually less than that for sequencing.

There are usually three possible reasons for this issue. First, it could be due to a small sample volume and low EV yield, resulting in such a outcome. Second, if an opaque ultracentrifuge tube is used, it is generally difficult to observe significant precipitation since EV production is typically small. Third, some brands of centrifuge tubes have low adhesion to the tube walls, causing the precipitation to slide down shortly after ultracentrifugation. Therefore, it is important to collect the samples promptly after centrifugation.

In ideal conditions, extracellular vesicles should exhibit a distinct cup-shaped morphology with a slightly brighter rim in negative staining electron microscopy results. Spherical structures without membranes are more likely to be lipoprotein particles (LDL) or aggregated proteins.

According to methodological literature, once the cell culture supernatant is collected, it should be processed promptly to isolate the extracellular vesicles. If the storage period is within two to three days, it can be kept at 4°C, although prolonged storage of the culture medium is not recommended. Extracellular vesicle samples should ideally be used directly after isolation and purification without undergoing freeze-thaw cycles. If longer-term storage exceeding seven days is necessary, it is recommended to store them at -80°C.

These protein markers were initially discovered through the purification of EVs followed by mass spectrometry analysis, which revealed their presence in high abundance within EVs. CD63, CD9, and CD81 are members of the tetraspanin protein family, which directly participate in the sorting of cargo within EVs. TSG101 is a protein associated with the ESCRT complex, and ALIX is involved in the process of vesicle scission from the limiting membrane, forming an independent membrane structure.

No, these marker proteins mainly involve the processes of vesicle formation and secretion. Most of them are membrane proteins or members of the ESCRT complex and its associated proteins, which are present in cells and localized near membrane structures. However, because these molecules are involved in the formation of vesicles, they are more abundant in vesicles compared to cells.

Currently, it is challenging to obtain highly pure and homogeneous extracellular vesicle samples using available isolation methods. Moreover, no single experiment can definitively confirm the presence of extracellular vesicles. For example, methods like ultracentrifugation and gel filtration cannot effectively remove lipoprotein particles from serum, even with density gradient centrifugation. Therefore, three identification techniques are used to confirm that the purified product is extracellular vesicles.

Regarding the three methods, electron microscopy can visualize morphology, but similar structures such as mycoplasma, ferritin aggregates, and lysosomes can also have similar appearances. NTA calculates the size distribution and quantity of particle populations based on the particle's effect on the light path, but it cannot reflect particle morphology. It classifies both extracellular vesicles and nanoparticles as particles, making it unable to differentiate between them. WB detects marker protein expression in extracellular vesicles, but these marker proteins are not exclusive to extracellular vesicles. Proteins such as CD63 and ALIX are present in cells and can also be abundant in cell fragments, lysosomes, and other intracellular compartments. Therefore, these three methods are used together for mutual validation.

Currently, there are three main methods for quantifying extracellular vesicles:

• Protein quantification: Using methods like BCA, the isolated extracellular vesicles are quantified by measuring the protein content to reflect the amount of extracellular vesicles.
• Particle counting: Using a nanoparticle counter, the number of extracellular vesicle particles is calculated to determine the quantity of extracellular vesicles.
• ELISA: Quantification of extracellular vesicles can be done using ELISA with specific surface markers, such as CD63, CD81, etc.

When choosing a quantification method, any one of the three can be selected, but consistency should be maintained throughout the research. BCA quantification can be affected by contaminating proteins, nanoparticle counting has inherent errors, and ELISA analysis of extracellular vesicle proteins may also detect them in other membrane structures and in the free system.