Exosome is a membrane vesicle released to extracellular matrix upon integration of cellular multivesicular body (MVB) and cell membrane, with a diameter of about 40~100 nm. Exosome naturally exists in body fluid, including blood, saliva, urine and breast milk, and the released exosome enters into blood, saliva, urine, breast milk and other body fluids, and reaches other cells and tissues through circulation system, generating remote regulatory effects. Exosome carries multiple kinds of proteins, lipids, DNA, RNA and other important information, which not only plays an important role in cell-to-cell material and information transmission, but also has the potential to serve as a biomarker for many early-stage diseases.

1. Extraction of exosome

Exosome exists in various body fluids in human or animals, because exosome is an MVB with a lipid bilayer membrane structure, which is formed by the invagination of the cell membrane, distributing in extracellular matrix.

The extraction methods of exosome mainly consist of differential centrifugation, density gradient centrifugation, magnetic bead immunization, ultrafiltration, polymer precipitation, and molecular exclusion.

The basic steps for exosome extraction include refreshing cell culture medium, collecting and incubating supernatant, ultracentrifugation for extraction, and washing. The specific processes are described below:

(1) Cell culture: selecting appropriate cell lines, culturing them to logarithm growth, and ensuring best cell state.

(2) Refreshing culture medium: in the later stage of cell culture, changing culture medium into exogenous serum-free medium, contributive to reducing background noise and elevating the purity of exosome.

(3) Collecting culture supernatant: collecting culture medium, transferring it to centrifuging tube, and performing centrifugation (generally 300~400 g, 10 min) to remove cells and cell debris.

(4) Ultracentrifugation for extraction: transferring supernatant to new centrifuging tubes, and conducting ultra-centrifugation (100.000 g, 70 min).

(5) Washing: resuspending precipitated exosome, and performing ultracentrifugation again to remove impurities generally in phosphate buffer saline (PBS).

2. Identification of exosome

Due to the limitation of separation and purification methods, it is hard to obtain exosome with high purity and strong singularity. Thus, subsequent multiple experiments are required to evaluate the purity and completeness of exosome samples.

International Society for Extracellular Vesicles (ISEV) proposed in 2014 three markers for identifying exosome: transmission electron microscope (TEM), Nanoparticle Tracking Analysis (NTA), and Western Blot (WB).

(1) Electron microscope imaging: TEM is a method identifying exosome from morphology and size aspects, which through re-staining methods can be applied to directly observe the morphological structure and size of single exosome. However, TEM has background interference and the inabilities to distinguish exosome from particles with similar morphology.

(2) Particle size measurement: it can perceive the existence of particles through optical principles, and detect the group feature of exosome quantify and diameter on the whole, which has the advantages of being accurate, fast and having good repeatability and is a good supplement for existing exosome identification methods. However, it cannot confirm the real existence and completeness of exosome and is not suitable for detection of complex samples.

(3) WB determination: based on the principle of the specific combination of antigens and antibodies, it can examine marker proteins of exosome, which can prove the existence of exosome components from the aspects of proteins to identify exosome. The commonly used exosome marker proteins include CD63, TSG101 and Alix.

3. Application of exosome

(1) Exosome as a drug carrier: achieving targeted drug delivery. Exosomes have the ability to carry “cargo” and deliver it to target cells, which can be used for intercellular communication, especially for remote communication. Exosome as a drug transport carrier, has unique advantages of low immunogenicity, high transport efficiency, strong stability, strong targeting ability, and the ability to cross the blood-brain barrier. Exosome is also appropriate for transmitting different chemical materials, proteins, nucleic acid, etc. Buller B et al. injected miR-146 loaded exosomes into the tumor site of mice with keratinocyte tumors and found that miR-NAS effectively inhibits tumor growth.

(2) Molecular markers for disease diagnosis: different kinds of cells in different diseases can all secret miRNAS/RNAS-containing exosome, making it possible to isolate exosomes from patients’ body fluids for disease diagnosis, especially in tumor diagnosis. In addition, exosome can be applied for diagnosing non-tumor diseases. Welker MW et al. discovered that serum-derived exosome CD81 may act as a potential protein marker for the diagnosis of chronic hepatitis C.

(3) Promoting tissue repair and regeneration: Recent studies have found that stem cell-derived exosomes are an important way for stem cell paracrine activity, which can mediate their promotion of tissue regeneration. Chen et al. found that the combination therapy of ADSCs-exo can reduce the infarct size of acute ischemic stroke in rats and promote neurological function recovery. ADSCs-exo has no disadvantages of immune rejection and ethical issues in stem cell therapy, and has advantages such as high stability, easy storage, no need for proliferation, and easy quantitative use. Compared with single cytokines, it has higher safety and greater tissue regeneration potential.