(B-53) Evaluating the Matrix-Bound Vesicles Extraction Process from Blood-Rich Tissue vs Cartilage Tissue

Introduction:: Extracellular vesicles (EVs) are small, lipid bilayer-enclosed particles released by cells into the extracellular environment that contain various biomolecules, including proteins, lipids, RNA, and DNA, making them a promising tool for a variety of biotechnological applications. A novel type of EV called Matrix-bound vesicles (MBVs) has been reported, which are EVs embedded within the extracellular matrix (ECM) fibers. Porcine tissues have been identified as a suitable source for isolating MBVs.

Characterization of MVBs is a critical step in the research and development of biotechnological applications based on MVBs. This study describes a differential digestion process (enzymatic vs. non-enzymatic) for obtaining MBVs from porcine thyroid cartilage and liver tissue. We hypothesize that the non-enzymatic protocol will yield a lower number of particles per milligram of ECM processed but will preserve transmembrane markers in the MBVs, making them suitable for developing biosensing tools. The nanoparticle tracking analysis (NTA) technique is a widely used tool for characterizing MVBs, based on the visualization of individual particles in solution using microscopy and the analysis of their Brownian motion. The technique provides information on the size distribution of the particles and their concentration.


Materials and Methods:: To obtain decellularized tissue (carti-dECM or lvr-dECM), porcine thyroid cartilage and liver tissue were dissected and cut into small pieces (3x3 mm) and washed with saline (PBS) to remove blood and other tissues. The tissue was then decellularized using a bioreactor through an automated process with 4% Sodium deoxycholate, followed by washing with PBS and MiliQ. The dsDNA content in the dECMs was measured to confirm decellularization. Subsequently, the dECM was digested to release the MVBs and centrifuged to separate the MVBs from the remaining undigested ECM fibers. The MVBs were then purified using an affinity-based size exclusion chromatography method with a qEV Column (IZON). The size distribution of the MVBs was determined by dynamic light scattered measurement and the concentration of MVBs was determined via nanoparticle tracking analysis (NTA).


Results, Conclusions, and Discussions:: The results of the study showed a high removal efficiency of cells and genetic material, with a significant reduction of dsDNA to 1.2 ug dsDNA/mg ECM. Nanoparticle tracking analysis (NTA) revealed a concentration of 1E9+/-2.96E7 particles per milliliter and an average size distribution of 100 to 200 nm for the MBVs. Purification using size exclusion chromatography yielded a fraction enriched in MVBs. Immunoblotting assays showed the detection of CD144 as an endothelial marker in lvr-MBVs but limited detection in carti-MBVs, suggesting that the proposed decellularization and purification protocol effectively obtains tissue-specific MBVs. These findings have important implications for future studies in therapeutic and diagnostic applications. Ongoing efforts are being made to determine MBV isolation yields under different conditions.


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