Isolation and Characterization of Human Adipose Derived Mesenchymal Stromal Cells and the Small Extracellular Vesicles They Produce

Abstract

Mesenchymal stromal cells (MSCs) are unspecialized cells that can be isolated in small quantities from adult tissues (e.g. fat, bone marrow). They can be cultured to increase their numbers to clinically relevant numbers and then implanted into damaged or diseased tissue to upregulate repair. Evidence now suggests that the therapeutic qualities of MSCs may be due, at least in part, to the small extracellular vesicles (sEVs) they release during culture. sEVs are non-living, membrane-bound nanoparticles that, upon release, migrate to target cells where they deliver bioactive cargo to elicit specific biological responses, such as tissue repair. Given their non-living nature, sEV-based therapies have fewer translational roadblocks compared to MSC implantation. However, the production of functionally tailored sEV populations for clinical purposes will first require the development of standardized protocols for MSC isolation and culture. During this project, a comprehensive protocol was developed to isolate cells from fat tissue and was conducted using both serum-containing and serum-free culture medium. Serum is a commonly used animal-derived component of culture medium, despite evidence that it alters cell behaviour and contains contaminating sEVs. A combination of techniques based on the literature was used to characterize cell and nanoparticle populations, which demonstrated that the populations possessed MSC- and sEV-associated characteristics, respectively, in both serum-containing and serum-free media. However, the extent to which they exhibited these characteristics in each type of medium differed. The protocols developed during this project will allow researchers to reproducibly generate MSC-derived sEVs, thereby bringing the field closer to the widespread implementation of sEV-based therapies.