Mesenchymal stem cells (MSCs) are being extensively investigated for their potential in tissue engineering and regenerative medicine. However, recent evidence suggests that the beneficial effects of MSCs may be manifest by their released extracellular vesicles (EVs); typically not requiring the administration of MSCs. This evidence, predominantly from pre-clinical in vitro and in vivo studies, suggests that MSC-EVs may exhibit substantial therapeutic properties in many pathophysiological conditions, potentially restoring an extensive range of damaged or diseased tissues and organs. These benefits of MSC EVs are apparently found, regardless of the anatomical or body fluid origin of the MSCs (and include e.g., bone marrow, adipose tissue, umbilical cord, urine, etc). Furthermore, early indications suggest that the favourable effects of MSC-EVs could be further enhanced by modifying the way in which the donor MSCs are cultured (for example, in hypoxic compared to normoxic conditions, in 3D compared to 2D culture formats) and/or if the EVs are subsequently bio-engineered (for example, loaded with specific cargo). So far, few human clinical trials of MSC-EVs have been conducted and questions remain unanswered on whether the heterogeneous population of EVs is beneficial or some specific sub-populations, how best we can culture and scale-up MSC-EV production and isolation for clinical utility, and in what format they should be administered. However, as reviewed here, there is now substantial evidence supporting the use of MSC-EVs in tissue engineering and regenerative medicine and further research to establish how best to exploit this approach for societal and economic benefit is warranted.

Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs’ CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.