Exosomes refer to discoid vesicles with a diameter of 40-100 nm. First discovered in the reticulocytes of rats and sheep by Harding et al. and Pan et al. respectively in 1983, they were officially named "Exosome" by Johnstone in 1987. Exosomes contain a variety of substances such as proteins, mRNA and microRNA. They can not only be used for disease diagnosis and condition monitoring, but also play roles in cell therapy, drug delivery, medical cosmetology and other fields. Hence, research on exosomes has remained a hot topic, and relevant papers are frequently published in top journals such as Science and Nature.
Formation Process of Exosomes
Studies have shown that all types of cells can secrete exosomes, including immune cells, epithelial cells, embryonic stem cells, mesenchymal cells and cancer cells. The formation process of exosomes is as follows:
- First, the cell membrane invaginates to form cup-shaped early sorting endosomes (ESE), which mainly contain exosomal proteins, membrane proteins and recycled exosomes.
- Then, the membrane of ESE invaginates inward and encapsulates cytoplasmic proteins, nucleic acids, metabolites and other substances inside ESE, forming late sorting endosomes (LSE) containing intraluminal vesicles.
- Finally, LSE matures into multivesicular bodies (MVBs). Some MVBs fuse with autophagosomes or lysosomes and undergo degradation, while other MVBs fuse with the plasma membrane and release intraluminal vesicles outside the cell. These intraluminal vesicles released outside the cell are the final exosomes.
Protein Types of Exosomes
Although the components of exosomes secreted by different types of cells or the same type of cells under different conditions vary, exosomes share certain characteristics in protein types, which are mainly classified into the following categories:
- Integral exosomal membrane proteins: Mainly include tetraspanins (CD81, CD82, CD37, CD63) and their chaperones (MHC II, IGSF8, ICAM-1, SDC1-4, Integrins, etc.), as well as membrane signaling proteins (EGFR, c-Kit, GPCRs, Notch receptors, T cell receptors, cytokine receptors, etc.).
- Lipid-anchored outer membrane proteins: Include C-terminal glycosylphosphatidylinositol (GPI)-anchored proteins (CD39, CD73, Juno, CD55, CD59, glypican-1, PrP SC) and Hedgehog morphogens, etc.
- Lipid-anchored inner membrane proteins: Include acylated small GTPases (Rabs, Ras, Rho, etc.), myristoylated signal kinases (Src) and palmitoylated membrane proteins, etc.
- Peripheral surface proteins: Include Wnt proteins and their receptors/chaperones, GPR177, BMPs, TGF β, TNF, TNF-related apoptosis-inducing ligand, FAS ligand, cytokines and ECM proteins (Fibronectin, Tenascin C, ECM1, etc.).
- Intra and peripheral membrane proteins: Include cytoskeleton-linking proteins (ERMs) and their ligands (EBP50, CD44, CD43, IGSF8, PTGFRN, etc.), Alix and its ligands (TGS101, ESCRTs 0-4), and molecular chaperone HSPs (HSP70, Hsp40/DnaJ, Hsp90, Hsp20, Hsp27, α/β-crystallins).
- Exosomal enzymes: Include CD39, CD73, phosphatases, pyrophosphatases, calcium-binding adaptor proteins, phosphate transporters, RNA editing enzymes, lipases, proteases, glycosyltransferases, glycosidases and metabolic enzymes, etc.
- Soluble proteins and inclusions: CD81, CD9, CD63 and other heterogeneous proteins.
Extraction Methods of Exosomes
There are various exosome extraction methods at present, including differential centrifugation, density gradient centrifugation, kit-based extraction, size exclusion chromatography, ultrafiltration, polymer precipitation, immunoaffinity isolation and pressure sieving. Among them, differential centrifugation is still one of the most common exosome separation technologies.
The principles, advantages and disadvantages of different extraction methods are shown in the table below:
表格
| Method | Principle | Advantages | Disadvantages |
|---|---|---|---|
| Differential centrifugation/Density gradient centrifugation | Purify and obtain exosomes step by step using the action of different centrifugal forces | Suitable for large-volume samples, low cost | Time-consuming, low yield |
| Polymer precipitation | Precipitate exosomes with hydrophobic polymers | No special equipment required, simple operation | Polymer particles mixed in products, impurity proteins introduced, yield related to polymers |
| Ultrafiltration | Centrifugal filtration with ultrafiltration membranes with a pore size of less than 150 nm | Short time-consuming, no volume limit, few impurity proteins | Easy to cause exosome loss and damage, yield greatly affected by temperature, viscosity and concentration |
| Immunoaffinity isolation | Precipitate exosomes by specific adsorption of exosomal surface antigens with magnetic beads | High selectivity, able to screen specific types of exosomes | Antibody impurities contained, volume limit exists, unable to screen all exosome subpopulations |
| Size exclusion chromatography | Separate particles based on the different elution times of particles with different particle sizes through gel columns | High purity, few impurities, short time-consuming, low cost | Samples need dilution, low separation volume and flux |
| Pressure sieving | Allow exosomes to pass through membranes by pressure or electric field | High exosome purity | - |
Identification Methods of Exosomes
Morphological Characterization
Observe the morphology of exosomes using transmission electron microscopy (TEM, a common method), scanning electron microscopy (SEM), cryo-electron microscopy (Cryo EM) or atomic force microscopy (AFM), etc.
Tips: (1) At least two methods should be used for morphological identification; (2) Exosomes are round with a diameter of 40-150 nm.
Particle Size Distribution Characterization
Analyze the diameter of exosomes using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA, a common method) and tunable resistive pulse sensing (TRPS), etc.
Protein Marker Analysis
According to the recommendation of the International Society for Extracellular Vesicles (ISEV), at least three types of proteins (two positive and one negative) should be detected for exosome analysis, including:
- Transmembrane or GPI-anchored proteins (positive): CD63, CD9, CD81, etc.;
- Internal exosomal proteins (positive): TSG101, HSP70 and Alix, etc.;
- Other organelle or cell structure proteins (negative): Histone, LaminA and Calnexin, etc.
Tips: CD63, CD9, CD81, TSG101, HSP70 and Alix are the most commonly used exosome markers.
Preservation Methods of Exosomes
According to the research by Cheng et al., storing exosomes in a preservation solution with a pH of 7.0 at -80℃ can maintain the activity and integrity of exosomes to the greatest extent. In addition, avoiding repeated freeze-thawing as much as possible can also maintain the activity and integrity of exosomes.