No abstract

In the recent years, the possibility of utilizing extracellular vesicles for drug delivery purposes has been investigated in various models, suggesting that these vesicles may have such potential. In addition to the choice of donor cell type for vesicle production, a major obstacle still exists with respect of loading the extracellular vesicles efficiently with the drug of choice. One of the proposed solutions to this problem has been drug loading by electroporation, where small pores are created in the membrane of the extracellular vesicles, hereby allowing for free diffusion of the drug compound into the interior of the vesicle. We investigated the utility of adiposederived stem cells (ASCs) as an efficient exosome donor cell type with a particular focus on the treatment of glioblastoma multiforme (GBM). In addition, we evaluated electroporation-induced effects on the ASC exosomes with respect to their endogenous potential of stimulating GBM proliferation, and morphological changes to single and multiple ASC exosomes. We found that electroporation does not change the endogenous stimulatory capacity of ASC exosomes on GBM cell proliferation, but mediates adverse morphological changes including aggregation of the exosomes. In order to address this issue, we have successfully optimized the use of a trehalose-containing buffer system as a way of maintaining the structural integrity of the exosomes.

The potential therapeutic utility of extracellular vesicles (EVs) has spawned an interest into a scalable production, where the quantity and purity of EV samples is sufficient for clinical applications. EVs can be isolated using several different protocols; however, these isolation protocols and the subsequent methods of quantifying the resulting EV yield have not been sufficiently standardized. Therefore, the possibility of comparing different studies with respect to these parameters is limited. In this review, we have presented factors that might influence the yield and function of EVs from cell culture supernatants. The methods of isolation, downstream quantification, and culture conditions of the EV producing cells have been discussed. In order to examine the inter-study coherency of EV yields, 259 studies were initially screened, and 46 studies were included for extensive downstream analysis of EV yields where information pertaining to the isolation protocols and quantification methods was obtained from each study. Several other factors influencing yield were compared, such as cell type producing EVs, cell confluence level, and cell stimulation. In conclusion, various factors may impact the resulting EV yield, including technical aspects such as EV isolation and quantification procedures, and biological aspects such as cell type and culture conditions. The reflections presented in this review might aid in future standardization of the workflow in EV research.