Background: Matrix-bound extracellular vesicles (EVs) are key local mediators of cardiovascular calcification. However, isolation and characterization of EVs from fibrocalcific tissue is challenging. Here, we sought to establish a streamlined methodology to isolate tissue EVs from frozen specimens.

Methods: To isolate EVs, frozen calcified human aortic valves underwent collagenase digestion, serial ultra-centrifugation, and density gradient fractionation. The protocol was halted and frozen at three centrifugation steps and compared to fresh-frozen tissue with no stoppage (N=2/group). Isolations were characterized using transmission electron miscopy (TEM), nanoparticle tracking analysis (NTA), and proteomics.

Results: EVs isolated from later freezing stop-points had more particles of EV-like size (2.56×10¹¹±1.03 x10¹¹ vs 2.36×10¹²±1.34 x10¹¹, p<0.001). TEM visualized different morphological and contaminant profiles at each stop-point, including collagen and lipid particles. Freezing later in the isolation protocol improved the similarity of proteins identified in the samples (57% vs 80%) compared to frozen control. Confirmatory EV signature was characterized by the identification of 14 key vesicular cargo and surface markers.

Conclusions: EVs that were isolated after freezing later in the protocol had the most similarity to fresh frozen tissue. Optimization of this protocol will facilitate the integration of EV isolation from fibrocalcific tissues and enable ex vivo EV studies.