Exosomes are extracellular vesicles (EVs) produced in endosomes of eukaryotic cells, which are secreted by different types of cells. They play a key role in intercellular communication of various normal and pathological functions. As potential disease biomarkers and therapeutic agents, exosomes are attractive in biotechnology and biomedical research. The key to achieving this potential is the ability to produce high-quality exosomes.
Fig 1. The biogenesis pathways and biochemical composition of A) exosomes, B) microvesicles, and C) apoptotic bodies. (Lai JJ, et al. 2022)
Exosome Processing and Isolation
The important factors to be considered in exosome processing and isolation include sample types and the choice of separation technology, which will affect the quality and characteristics of the products. Common techniques include ultracentrifugation, density gradient separation, and size-exclusion chromatography.
Table 1. Exosome processing techniques (Lai JJ, et al. 2022)
| Process |
Time [min] |
Sample types |
Volume (mL) |
Advantages |
Disadvantages |
| Ultracentrifugation |
140–600 |
CCM, urine |
≤25 |
Good purity for clinical treatment applications and proteomic studies |
Impurities (e.g., protein aggregates)
Expensive instrument
Complex procedure
Repetitive steps damage isolated vesicles |
| Ultrafiltration |
130 |
CCM, urine |
≤15 |
High throughput
Wide range of sample volume
Simple procedure |
Low purity
Reduced yield by filter clogging
Not suitable for plasma |
| Precipitation |
30–120 or overnight |
CCM, plasma |
≤10 |
High yield
High throughput
Simple procedure |
Low purity (e.g., polymer contamination)
Not suitable for plasma |
| Immunoaffinity capture |
240 |
CCM |
0.5–3 |
High purity for proteomic analyses
High selectivity |
Low yield
Low sample volume
Expensive
Extra elution step |
| Size-exclusion chromatography |
15 |
CCM |
0.5–1.5 |
Higher purity than precipitation
Low required volume
Versatile for various specimen types
Preserves vesicle integrity |
Protein contamination
Low yield
Expensive instruments and column
Complex procedure
Dilution is required for viscous samples |
Exosome Characterization
Characterizing exosomes is essential for understanding their composition and function. Here are some techniques commonly used for exosome characterization:
Electron Microscopy: Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) allow researchers to visualize exosomes and assess their morphology and size.
Nanoparticle Tracking Analysis (NTA): NTA provides quantitative data on the size distribution and concentration of exosomes in a sample.
Flow Cytometry: Flow cytometry can be used to analyze exosomes for surface markers and assess their purity.
RNA and DNA Analysis: Techniques like PCR and RNA sequencing are employed to analyze the genetic material carried by exosomes.
In conclusion, the research and development of exosomes is different from other biologics such as proteins, which is lack of standardized Good Manufacturing Practices for their processing and characterization. It is necessary to find appropriate exocrine production, separation, downstream purification, and analysis, so that it can be used as a safer and more effective therapeutic agent in diagnostic applications.
Reference
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Lai JJ, Chau ZL, Chen SY, et al. Exosome Processing and Characterization Approaches for Research and Technology Development. Adv Sci (Weinh). 2022;9(15):e2103222.
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