Exosome Quality Control: How to Do It?

Ensuring standardized and scalable production of exosomes, along with a robust quality management system, is essential to advancing their research applications and clinical translation. This is not only related to the stability and reliability of exosome products, but also an important foundation for ensuring their security and effectiveness. At present, for the comprehensive characterization of exosome quality, the industry has formed a set of systematic measurement standards. which are mainly focused on a range of core aspects, including but not limited to particle size analysis, concentration, surface markers, microbial and mycoplasma detection, and so on. The following are examples of commonly used exosome quality control systems and assays.

Exosome Particle Size and Concentration

Currently, the main methods to analyze the particle size and concentration of exosomes are FlowNanoAnalyzer and NTA (Nanoparticle Tracking Analysis). These methods enable precise measurement of exosome size and provide size distribution profiles.

Fig. 1. Particle concentration and size distribution of exosomes measured by NTA (Zhu T, Sun J, et al., 2022).

Positive Rates of Protein Markers (CD63/CD81/CD9)

Exosomes typically express markers such as CD81, CD63, and CD9, which produce strong fluorescence when incubated with exosomes using fluorescently labeled CD81/CD63/CD9 antibodies that bind to the exosomes, then detected by flow cytometry. This detection technique is quite rapid and suitable for high throughput screening.

Fig. 2. Flow cytometry showed three positive markers for exosomes: CD81, CD63 and CD9 (Pourhadi M, Zali H, et al., 2024).

Exosome Morphology

A well-established way to identify the morphology of exosomes is transmission electron microscopy (TEM), which can also identify exosomes by morphology and size and generate TEM images. In contrast to negative staining, we can directly examine the shape, structure and size of individual exosomes through a powerful high-power microscope.

Fig. 3. Transmission electron microscopy of native exosomes (Aguilera-Rojas M, Badewien-Rentzsch B, et al., 2018).

Sterility Testing

Sterility testing ensures that no live bacteria, fungi or viruses get in contact with exosome samples. The test is usually made by culture medium culture method. It is vital that the test is handled under aseptic conditions. Rapid techniques like PCR or enzyme-linked detection kits can also identify specific bacterial or viral DNA/RNA.

Mycoplasma Testing

Mycoplasma contamination is typically detected using specialized test kits, where amplification of conserved mycoplasma DNA sequences induces a visible color change in the reaction solution. Other methods include culture and DNA staining techniques.

Pathogenic Factor Screening

The test for intra- and extracellular pathogenic factors should be negative for HIV antibody, HBsAg, HCV antibody, TP antibody, CMV-IgM, HTLV antibody, HPV antibody, HHV antibody, EBV antibody, Furthermore, nucleic acid tests for HCV, HBV, and HIV should also be negative. Generally, ELISA for antibody level and nucleic acid test for viral nucleic acid are used.

Endotoxin Testing

Common endotoxin detection methods include the Limulus Amebocyte Lysate (LAL) test, which detects endotoxins through a colorimetric or turbidity-based gelation reaction. There are alternative methods such as recombinant factor C tests or fluorescence polarization techniques. For example, the endotoxin standard for mesenchymal stem cells requires a value no greater than 0.5 EU/mL.

Abnormal Immune Response Testing

To assess abnormal immune responses, exosome samples should demonstrate no abnormal lymphocyte proliferation. This is measured using CFSE-labeled lymphocyte proliferation inhibition assays, with flow cytometry to assess lymphocyte proliferation rates.

References

1. Zhu T, Sun J, et al. Plasma Exosomes from Children with Atopic Dermatitis May Promote Apoptosis of Keratinocytes and Secretion of Inflammatory Factors in vitro. Clin Cosmet Investig Dermatol. 2022. 15:1909-1917.
2. Pourhadi M, Zali H, et al. Restoring Synaptic Function: How Intranasal Delivery of 3D-Cultured hUSSC Exosomes Improve Learning and Memory Deficits in Alzheimer's Disease. Mol Neurobiol. 2024. 61(6):3724-3741.
3. Aguilera-Rojas M, Badewien-Rentzsch B, et al. Exploration of serum- and cell culture-derived exosomes from dogs. BMC Vet Res. 2018. 14(1):179.

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