Extracellular vesicles (EVs) have been widely investigated as potential vehicles for targeted therapeutic compound delivery and regenerative medicine. Natural EVs are released by cells and are considered an additional mechanism facilitating communication between cells. Through this system cells are capable of exchanging DNA, RNA, proteins, and lipids to neighboring and remote sites in the body. The main subtypes of EVs are exosomes, microvesicles and apoptotic bodies, which are of various sizes and have different secretion pathways. A thorough understanding of EVs gained through further research and analysis of EVs will further expand the clinical application of these particles.
Exosomes are a type of EV that have have gained attention of the scientific community due to their unique characteristics, including their ability to target different tissues, their capacity to overcome biological barriers, and compared to the other gene therapy vectors they can evade detection from the host immune system, providing a safer route for the carried therapeutic material to reach the target.
It is highly important to assess the quality of EVs throughout the manufacturing process. TEM analysis provides key data for monitoring and comparing the effect of altering certain conditions on the stability, integrity, and purity of the produced EVs. TEM analysis provides information on the following parameters:
Cryogenic transmission electron microscopy (cryoTEM) has been used as one of the most important analytical tools to characterize the properties of EVs and, more specifically, exosomes. When imaged with cryoTEM, exosomes containing genetic material such as lipids, proteins, or nuclear acids, reveal different pixel intensitites in their interior compared to other particles in solution. This characteristic can be used to identify distinct morphological features in different exosomes, and can also be used to identify exosomes in a sample that contains a plethora of similar particles.
Figure 1 – Representative cryoTEM images of exosomes; engineered exosomes (left) and purified exosomes from human serum (right).
Figure 1 displays two types of exosome samples. The sample shown in the left column is comprised of engineered exosomes of various sizes and morphology. Particles displaying a dark internal density, as well as multilamellar and multiparticle types are observed. The sample shown in the right column contains purified exosomes derived from human serum of healthy donors. These particles display a higher level of pleomorphism than the particles in the left column. In addition, the image background is noisier, indicating that the sample has a lower purity.
In collaboration with our clients, we can define the particle classes identified in our client’s product. Using this information, Vironova’s proprietary software, VAS, determines the particle class distribution for the sample, providing statistical data that can be correlated with the biological significance of the product, and that may also help to guide and inform further processing decisions. For further information on this type of analysis and its suitability for your product, please contact us.
A unique characteristic of the cryoTEM sample preparation procedure is that it preserves the native conformation of EVs, making it one of the few techniques that can reliably measure both the size and internal volume of exosomes.
Circularity and size distribution analyses respectively provide information about the shape and size of the exosomes in a sample, while an internal volume analysis provides a profile of loading capacity (see Figure 2).
Figure 2 - Representative histogram showing the size distribution (left) and internal volume distribution (right), as determined from semi-automated detection.
Negative stain transmission electron microscopy (nsTEM) is a technique which involves the application of a heavy metal salt solution to a sample for particle embedding and enhanced image contrast. As with cryoTEM, nsTEM offers a suitable route for imaging particles to gain an understanding of the particle size distribution. Moreover, nsTEM is a valuable technique for inspecting the purity and integrity of exosome samples (see Figure 3). A noisy image background may indicate a low degree of purity of a sample, while broken or aggregated particles may highlight issues with sample integrity. With nsTEM, this critical information can be discerned and correlated to product quality, informing further processing decisions.
Figure 3 - Representative nsTEM images of exosome samples with broken particles and aggregates.
What is the optimal concentration of exosomes required for TEM analysis?
A sample concentration of 5x1,011 particles/mL (measured by Nanoparticle Tracking Analysis) has shown to be represented in cryoTEM images with typically 3 to 4 EVs per image. The number particles per image is higher when the same sample is prepared for nsTEM. The sample preparation procedure may also be optimized for your own product to increase the on-grid concentration.
Does Vironova perform GMP-certified analysis on exosome samples?
Yes, Vironova can perform GMP-certified analysis on exosomes.
Can the purity of exosome samples be tracked between different purification steps?
Yes, it is possible to track the progress in quality of the product between purification stages with TEM analysis. Please contact us to discuss the type of analysis suitable for your samples.
Which biosafety levels are handled at Vironova's laboratory?
We can perform TEM analysis for samples up to biosafety level 2 at our GMP-certified laboratory in Stockholm.
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