Systematic review of factors influencing extracellular vesicle yield from cell cultures

Abstract

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.

Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes

“…In the present study, ASC exosomes were shown to be devoid of any proliferation-stimulating properties after 4 days on two different GBM cell lines, including a cancer stem cell line, underscoring their relevance with respect to safety in delivering therapeutics to GBM tumors. However, for ASC (or any other cell type's) exosomes to be used in any sort of therapy, one would also need to include a thorough assessment of how the cell culture conditions affect the production and characteristics of the secreted exosomes (Gudbergsson et al 2015). For example, several types of cell stress, such as hypoxia, cell activation and high glucose, are known to interfere with the released quantity of exosomes, but also with their composition of proteins and RNA, whereas the effects of other components of cell culture, like the surface structure of the culture dish, remain completely unknown (de Jong et al 2012;King et al 2012).…”

“…[ 150 nm) could also be detected. It should be noted that the distinction between different types of EVs cannot solely be based on their size distribution, as some level of diameter overlap will be expected (Gudbergsson et al 2015). The total yield of EVs from the CM was found to be high, averaging around 6 9 10 10 particles/T175 culture flask (Fig.…”

“…The CM was harvested after 24 h of incubation and exosomes were isolated by two different procedures. Implications of the choice of isolation protocol for obtaining exosomes and other extracellular vesicles (with respect to yield and purity) have recently been reviewed (Gudbergsson et al 2015).…”

“…Electroporation of ultracentrifuged exosomes in trehalose pulse medium does not modulate the size distribution Since sequential ultracentrifugation is known to yield exosome samples with higher purity than commercial isolation kits (Witwer et al 2013;Gudbergsson et al 2015), we wanted to assess whether such high purity could avoid the continued aggregation after electroporation. The ultracentrifuged ASC exosomes were resuspended in TPM, electroporated, and analysed by NTA.…”

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line

“…Ultracentrifugation-free methods for isolation of EVs have also been developed, including ultra-filtration, precipitation and immuno-affinity capture using specific antibodies. Although studies have investigated factors affecting the efficiencies of these methods [13–16], there is no study comparing the biological activities of the EVs isolated by these methods.…”