The vast majority of cells in the body can produce sEVs (13), which are found throughout our body. The sources of sEVs are mainly divided into two main categories, one is the direct extraction of sEVs from body fluids secreted by the human body, such as serum (14), lymph, cerebrospinal fluid (15), bile (16), plasma (17), urine (18), breast milk (19), saliva, etc., and the other is the extraction of sEVs from the supernatants of a variety of cell culture media (20). However, since natural body fluids, especially plasma, contain cellular debris, apoptotic vesicles, microvesicles, and plasma proteins, which are not easily separated from sEVs due to their overlapping sizes and biochemical properties, resulting in a low purity of the isolated sEVs (21). The urine has fewer interfering particles than the plasma, but a low concentration of sEVs (21). It’s obvious sEVs obtained from urine are more pure, but because of their low concentration, they require a larger volume than in plasma extraction to obtain the same mass.

In contrast, in vitro cell culture supernatants are easier to obtain and the quality of sEVs obtained is more stable, so most of the existing techniques are extracted and isolated from conditioned cell cultures (22, 23). Stem cells with high productivity have been used for the longest time for in vitro cell culture, with mesenchymal stem cells (MSCs) being the most used (24). Depending on the source, MSCs can be categorized into bone marrow MSCs, adipose MSCs, human umbilical MSCs, dental pulp MSCs and so on (25). Stem cells from different sources proliferate at different rates on their own and produce sEVs of varying quantity and quality (26), among which human umbilical MSCs produce the largest number of sEVs and the largest size (27), meanwhile, human umbilical MSCs are able to be stably cultured in serum-free medium (28), which effectively avoids the contamination by the impurities in serum in the subsequent isolation process, and is conducive to the large-scale production of sEVs.

The occurrence and secretion of cellular sEVs are influenced by multiple conditions. On the one hand, small molecules can influence sEV production and secretion. For example, thrombin pretreatment enhances the ability of MSCs to produce extracellular vesicles and the quality of extracellular vesicles is not affected (29). Adiponectin stimulates the production and secretion of sEVs by binding to T-cadherin on MSCs (30). N-methyldopa and norepinephrine can triple the sEV production of MSCs (31). Melatonin-treated sEVs enhance the regenerative potential of MSCs (32). In addition, external environments such as magnetic field (33), flow and stretch (34), ultrasound (35), PH (36), hypoxia (37), temperature (38), and light (39) affect the synthesis and release of sEVs.

To further obtain higher yields and quality of sEVs, Three dimensional culture systems are being used for sEVs production. The three dimensional culture improves sEVs production by increasing cell-cell-cell matrix interactions (40). Common three dimensional culture methods are hanging droplet culture and microporous array method, the hanging droplet culture is simple and easy to execute, but the yield is limited and there are limitations for sphere size adjustment, so mass production with hanging droplet culture will be time-consuming (41). The microporous array method inoculates cells into a series of small wells to which cell growth-promoting materials can be added to promote cell proliferation and sEV synthesis, In addition, the microporous array method is easier to produce three dimensional spheres than the droplet method and has a higher throughput (42), which means microporous array method more suitable for mass production. In addition, artificial sEVs with low cost, high yield and stable quality are expected to be a powerful alternative to natural sEVs (43).

Common isolation techniques include ultracentrifugation, ultrafiltration, immunoaffinity capture, size exclusion chromatography and precipitation.

Ultracentrifugation is the classical method for sEV isolation (44). At the same time, it is also the most widely used isolation technique (45). Ultracentrifugation is based on the separation of sEVs and impurities in the samples with different densities and sizes (46), the dead cells, cellular debris, and large extracellular vesicles in the samples are successively removed by different rotational speeds, and finally obtain the sEVs in the supernatant (45). Ultracentrifugation can process a large number of samples at one time and is easy to perform, but produces less pure and time-consuming sEVs because of the different subtypes of sEVs have overlapping density ranges (47). Formation of a density gradient medium with sucrose or iodixanol in combination with ultracentrifugation improves the purity of sEVs, but prolonged incubation with high sucrose concentrations impairs the structure of sEVs (48). In addition, the polymer density layer is expensive and not suitable for sEV scale-up.

Ultrafiltration is based on the isolation of different extracellular vesicles with different sizes, which can only pass through a series of semi-permeable membranes with defined pore sizes (49). However, since extracellular vesicles are deformable, vesicles that do not conform to the size can also be deformed to pass through the pore resulting in sEV impurity, so they are only used for preliminary isolation (50).

Immunoaffinity capture is an sEV isolation technique based on antigen-antibody interactions, in which immobilization of a specific antibody allows for specific binding of an antigen unique to the surface of the sEV, and thus capturing the sEVs (51). However, the overlap of antigens between different subpopulations and the difficulty of removing capture antibodies can affect later functional assay studies of sEVs, that not conducive to subsequent sEV research and applications.

Size exclusion chromatography (SEC) is also an isolation method based on sEV size, where large particles are unable to enter the gel pores and small sEVs are allowed to enter, which is a milder separation method that yields sEVs with higher purity and activity (52). However, the resolution of SEC decreases when the particles are close to or larger than the upper limit of the pore size, and so SEC is often used in conjunction with ultracentrifugation (53) and ultrafiltration (54) to improve the purity of sEVs.

Precipitation is sEVs under the action of polyethylene glycol usually, the solubility decreases leading to the precipitation of sEVs, and then sEVs can be obtained by low-speed centrifugation (55), which is easy to operate, does not require special equipment, and is conducive to the preparation of large-scale, but is prone to the introduction of impurities leading to the sEVs are not high purity (56).

As the demand for sEVs increased, a variety of commercial kits rapidly emerged. Kits principle is based on traditional sEV separation methods such as ultrafiltration, sedimentation and so on. Commercial kits do not require special equipment, with the advantages of simple operation and short time consuming, and can isolate sEVs from most common body fluids and cell culture supernatants (57). However, kits are expensive and cannot process a large number of samples at once, so they are not suitable for high-volume processing. Furthermore here are significant differences in the purity and quantity of sEVs collected by different kits (47). For example, the yield of sEVs obtained with the invitrogen kit is dozens of times more than that obtained with conventional ultracentrifugation, but the purity of the output sEVs is unsatisfactory, and the sEV isolation kit requires pre-separation before use, which makes the experimental process cumbersome (58).

Although there are many techniques for sEV isolation and purification, all of the above techniques have significant drawbacks and are not suitable for large-scale production of sEVs (Table 1). Microfluidics is an emerging method for sEV isolation, which is a technique for controlling fluids in micrometer-sized channels that relies on a range of sEV properties including immunoaffinity, density, and size, and it overcomes the limitations of traditional methods by offering advantages such as low cost, small size, speed, sensitivity, labeling-free, and high recoveries (57). It is expected to replace traditional methods in the future and play an important role in the industrialized mass production of sEVs in the future.

The EXODUS system separates and purifies sEVs by two coupled oscillators generating a dual-frequency transverse wave on the membrane, which produces sEVs at a rate, purity, and throughput that are far superior to the others (68). Asymmetric-flow field-flow fractionation technology (AF4) is label-free, fast, highly reproducible, highly recoverable and high resolution, which helps to separate different sEV isoforms (64). However, because AF4 separates based on particle size, it is not able to separate sEVs that have the same size, but are actually different. Double tangential flow size screening-based microfluidic chip greatly improves sEV recovery rate and purity. Its sEV recovery rate up to 77.8, acquired sEVs can be directly used for sEV analysis (65). Capture of sEVs by altering temperature was devised by Kenichi Nagase (71).

Whether it is the traditional separation technology or the microfluidic technology in the last two years, it has not fully achieved the ideal separation effect. Based on the properties of sEVs and downstream applications, it may be a useful idea to combine different isolation methods to get better separation effect. The combination of different techniques may offer the possibility of efficiently obtaining high-purity and high-yield sEVs.