Dos Reis Marques, R.; Sheth, M.; Salami, A. I.; Kongsomros, S.; Esfandiari, L.; Dewey, M. J.

Matrix-bound nanovesicles (MBVs) are a type of small extracellular vesicle (EV) embedded in the extracellular matrix (ECM) throughout the body. MBVs have been previously isolated from various tissues and in vitro-cultured cell sheets, demonstrating remarkable attributes in regenerative medicine. However, differences between MBVs and conditioned culture medium-derived EVs (liquid-EVs) have yet to be characterized, and the field currently lacks specific protein markers that can identify MBVs from other EV subtypes. Here, we isolate MBVs and liquid-EVs from bone marrow mesenchymal stem cell (MSC) sheets and define differences in size, protein, and zeta potential between these EVs. We show that there is a correlation between cell-driven ECM deposition and MBV and liquid-EV production. We also find that MBVs are smaller, contain less protein per particle, and possess lower zeta potential than liquid-EVs. Interestingly, MBVs also comprise a distinct tetraspanin profile compared to liquid-EVs, with MBVs containing more CD63 and little to no CD81. Finally, we define that CD63, LAMP1, Alix, ITG{beta}1, and GRP94 and their abundance, may be markers specifically used to identify MBVs from liquid-EVs. Our study paves the way for the characteristic differentiation between MBVs from liquid-EVs, elucidates their differences in biogenesis, and reveals a potential connection between EV and ECM production.