Exosomes

The Science of Exosomes

All Exovex products have undergone extensive molecular characterization including:
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The results of these tests and interpretation of the data is detailed below

Polymerase Chain Reaction (PCR)
Each specimen was tested against an extensive panel of pathogens including HIV, Hepatitis B, Hepatitis C, HTLV, Syphilis, CMV, West Nile virus, Zika virus. This panel exceeds FDA guidelines for communicable disease testing in human tissue.

 

No PCR amplification was observed for any of the pathogens tested. The results of this analysis confirm that the starting material is free of all communicable diseases tested.


Fluorescence Assisted Cell Sorting (FACS)
The cells were sorted using FACS to specifically select placental Mesenchymal Stem Cells prior to expansion. This level of analysis is not routinely performed prior to culture for Exosomes production. All cells expressed surface molecules CD105, CD90, CD73 and CD146 and CD44 and lacked expression of CD45, CD34, CD31 and HLA-DR [1]. This analysis confirms the presence of purified MSCs prior to culture resulted in a higher yield of exosomes nanoparticles.


Multidimensional Protein Identification Technology (MudPIT)
Two representative samples were submitted for MudPIT [2] analysis. Comparative analysis of each specimen demonstrated a high degree of inter-batch reproducibility. In Sample 1, 387 unique human proteins were identified from a total of 2665 peptide sequences detected. In Sample 2, 401 unique human proteins were identified from a total of 3914 peptide sequences detected.


All proteins detected in each sample were cross referenced against the UniProt database [3] for functional annotation to ascertain the potential efficacy of the Exovex samples by understanding the biological processes of their component proteins.


The predominant proteins identified in each sample tested are consistent between the different batches tested. Their function based on UniProt annotation is detailed in Table 1:

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The presence of multiple growth factors is of particular significance for the desired Exovex applications in anti-aging and wound healing. Based on the biological function of the constituent growth factor proteins detected in the Exovex samples, we would expect efficacy related to cellular proliferation and migration, epidermal rejuvenation, angiogenesis and cell- collagen interactions.

 
The details of the growth factors detected are listed in Table 2

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Table 2: The individual growth factors detected, including a link to their UniProt detailed functional characteristics.

 

In addition to growth factors, the proteins detected represent a broad spectrum of functional groups including collagen proteins, enzymes, Extracellular Matrix proteins,
Heat Shock Protein (HSP) molecular chaperones and cytokines.

 

The broad protein functional groups detected is represented in Figure 1

 

Figure 1: Breakdown of the protein functional groups detected in the MudPIT analysis

 

The ‘other’ category represents a multitude of different functional proteins including

The abundance and broad function of the proteins detected in both samples are indicative of a highly diverse final product with extensive therapeutic potential.

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Nanoparticle Tracking Analysis (NTA)
Two representative samples were submitted for NTA. Both samples demonstrated exceptional particle counts indicating the effectiveness of the upstream processes, in particular the proprietary culture media and the fluorescence assisted cell sorting, for producing a high potency final product.


Two complementary techniques for estimating the size and concentration of the particles in each sample were used [4]

The mean particle diameter and concentration in particles/mL are detailed in Table 3:

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Table 3: The mean particle size and concentration of each sample measured.

By comparison with other commercially available Exosomes products, Exovex has over 10-fold more particles/mL (Table 4)

 

The results of the NTA are indicative of a high potency final product and demonstrate the efficacy of the extensive upstream processes for producing a large quantity of Exosomes.

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Table 4: Concentration of commercially available Exosomes

Summary

The comprehensive molecular characterization performed on Exovex demonstrates a final product with a high concentration of Exosomes which contain a broad functional range of proteins. Further next generation sequencing based RNA characterization is currently underway to characterize the functional micro RNA populations present in each specimen.

 

In vitro testing of Exovex on epidermal monolayers in in progress to establish the functional characteristics obtained by applying a product with such promising molecular specifications.

 

Refferences

Dominici,M.etal.,“Minimalcriteriafordefiningmultipotentmesenchymalstromalcells. TheInternationalSocietyfor Cellular Therapy position statement.” Cytotherapy (2006) Vol. 8, No. 4, 315 –317

 

Schirmer, E.C. et al., “MudPIT: A powerful proteomics tool for discovery.” Discov Med. (2003) Oct;3(18):38-9.

 

TheUniProtConsortium“UniProt:aworldwidehubofproteinknowledge”NucleicAcidsRes. (2019)47:D506-515

 

Felipe, V. et al., “Critical Evaluationof Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates.” Pharm Res. 2010 May; 27(5):796–810.