The effect of culture conditions on the immunomodulatory properties of MSC-derived EVs

The microenvironment of cells, which determines their biological behavior, such as the packing and shedding of EVs, is the result of a multitude of physical, chemical and biological parameters. Thus, the biological functionalities of MSC-EVs are affected by the culture conditions of the parental cells. Especially, physiologic oxygen concentrations and physiologic cell-cell or cell-substrate interactions using 3D cell culture approaches have been shown to influence the number of secreted EVs, their surface marker signature, their cargo, and ultimately their biological function. Although it is clear that oxygen concentration and 3D cell culture during extracellular vesicles (EV) generation affect the secretion and cargo of MSC-EVs, it is not well understood if and how these parameters affect the immunomodulatory properties of MSC-EVs. Based on available scientific data and our preliminary results our central hypotheses are: 1) Major characteristics, such as number, size distribution, surface markers, and intracellular and membrane-bound proteins of MSC-EVs are affected by the culture conditions (2D vs. 3D) of MSCs. 2) Therefore, different culture conditions of MSCs affect the biological functions of MSC-derived EVs, in particular their procoagulant potential (exposure of TF) and their immunomodulatory properties (influence on monocyte subset distribution). To test these hypotheses, we will generate MSC-EV preparations from hydrogel, spheroid and flat surface culture from physioxic conditions and characterize these preparations towards their immunomodulatory properties and procoagulant potential. Here, we focus first on characterization of the EVs and EV cargo to understand how the abovementioned parameters affect formation and loading of MSC-EVs. Second, we will investigate the interplay between MSC-EVs from different culture conditions and cells of the adaptive and innate immune system. This will result in a detailed understanding of MSC-EV formation and loading and ultimately enable to manufacture MSC-EVs with defined immunomodulatory properties for the treatment of inflammatory conditions.