Extracellular vesicles (EVs) characterization

Application of extracellular vesicles (EVs) has been widely investigated as potential vehicles for targeted therapeutic delivery and regenerative medicine. Natural EVs are released by cells and are considered an additional mechanism of communication between cells. Through this system cells are capable of exchanging DNA, RNA, proteins, and lipids to neighboring and remote sites in the body. EVs comprise exosomes and microvesicles which have different sizes and secretion pathways. Knowledge that comes from the EVs analysis will shed light in the clinical application of these vesicles.

Exosomes (30–100 nm) are a type of EVs which have gained the attention from the scientific community due to their unique characteristics. These unique characteristics are: their ability to target different tissues, their capacity to overcome biological barriers, and compared to the other gene therapy vectors they can evade from the host immune system detection and degradation, providing a safer way for the carried therapeutic material to reach the target. 

What can be monitored in different manufacturing processes by using TEM

A close monitoring of the EVs upstream and downstream processing is needed. For this reason, the manufacturer’s main focus is guaranteeing that these vesicles are well characterized in terms of stability, purity, and integrity. TEM analysis provides comparable data when production conditions are changed in upstream and downstream processes or product formulations:

  • Product clustering and aggregating during engineering of the particles in the upstream and downstream processes.
  • Failure to remove host cell debris and impurities.
  • Presence of therapeutic material inside the exosomes (packaging analysis).
  • Changes of particle morphology or integrity due to changes in the process.
  • Morphology or integrity changes caused by modifications in the formulations.

CryoTEM will characterize internal morphology and product stability

CryoTEM has been used as one of the most important analytical tools to better characterize the properties of EVs and more specifically exosomes. Exosomes which carry different type of genetic materials, proteins, or lipids, will reveal different pixel intensities in their interior compared with other particles in solution, when imaged in cryoTEM. This morphological feature can be used to identify exosomes in a sample which contain plethora of similar particles. Exosomes can be engineered to express proteins on the surface and this can be directly observed by using cryoTEM.

Several CryoTEM images of exomes.

Figure 1 - CryoTEM images of exosomes.

In Figure 1, two types of samples were imaged. The first example (Figure 1, left column), the sample is composed by engineered exosomes, overall, the sample showed particles of different sizes and morphology. While particles showing an internal dark density were identified, also multiparticle and multilamellar particles were observed on grid.

The second example (Figure 1, right column), the sample contains purified exosomes from human serum (healthy donors), which showed a higher pleomorphism. The background of the second sample (right column) images is noisier which is correlated to the purity of the sample. Vironova’s proprietary software, VAS, provides statistical representation data of the distribution of this different classification of particles on the grid. This data is then correlated with the biological significance of the product, which is used to make informed decisions. This classification is done in collaboration with our clients, if you would like to know more about the potential of this analysis please contact us.

Circularity and Size distribution

The sample preparation for cryoTEM preserves the native conformation of the EVs which make it one of the few techniques that can reliably measure the size and the internal volume of the exosomes.

The circularity analysis provides information about shape of the exosomes and particle size distribution provides information about size of exosomes in the samples. The internal volume analysis provides a profile of loading capacity of the exosomes in the analyzed sample. The results of the analysis aids the characterization of exosomes in a sample (Figure 2).

Histograms of size distribution and internal volume of exosome sample.

Figure 2- The histograms illustrate the size distribution (left) and internal volume (right) of exosome sample obtained from semi-automated image analysis using VAS (about 1500 particles were detected for the analysis).

Negative staining will characterize the purity, stability, and size distribution

EVs vary in size between approximately 100 and 300 nm, this size distribution can also be described using negative stain transmission electron microscopy (nsTEM). Moreover, the integrity and purity of the exosomes samples can be reported using nsTEM, as shown in Figure 3. In these nsTEM images, the background of EVs samples is noisy due to debris of various sizes present in the sample. This characteristic background can be correlated to the degree of purity of the sample, which is used as a vital factor to take decisions on selecting efficient downstream processes.

Several images of exosomes samples imaged using the nsTEM technique.

Figure 3 - nsTEM images of exosomes samples, with broken particles and aggregates.


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