Investigating exosomes for regenerative medicine: development of purification techniques and novel analytical strategies for assessment of potency

Thomas Edmund Whittaker, Rosetrees Trust, Engineering And Physical Sciences Research Council, Natalie Stingelin, Molly Stevens
2021
No living cell has yet been reported that does not release extracellular vesicles (EVs). The last decade has seen an explosion of research into their properties as biomarkers, drug carriers, effectors of signalling and potential therapeutics. Within the field of regenerative medicine, EVs have recently been implicated in the therapeutic paracrine signalling of human mesenchymal stem cells (hMSCs) after transplantation. They appear to represent a promising cell-free therapy for myocardial
more » ... r myocardial infarction, stroke, and other diseases. However, effective purification of EVs without accompanying contamination from other cell-derived factors is challenging. There is debate within the field as to the fitness for purpose of several commonly-used isolation techniques. Cell signalling via EVs is not typically directly compared to signalling via conventional soluble factors, and there are no widely-adopted standards for assessing potency. Here, an effective EV isolation system was developed and validated to be capable of fully separating EVs from free protein. HMSC-EVs isolated with this system were found to be ineffective stimulants of in vitro assays of angiogenesis and wound healing. It was found that hMSC-EVs could only stimulate these assays when purified by a low-stringency technique or when used at very high concentrations. Ultrafiltration experiments similar to those that initially suggested the necessity of EVs in hMSC signalling were found to be prone to false positives. When evaluated in experiments that allowed direct quantitative comparison with non-EV factors produced by the same cells, non-EV factors were repeatedly found to be necessary and sufficient for hMSC paracrine stimulation of in vitro angiogenesis and wound healing. A conceptual framework for quantitative evaluation of the contribution of EVs to signalling processes is proposed.
doi:10.25560/89473 fatcat:xm2cnelfuvdoxe5sxlbzzgrnhm