Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use

Abstract

Extracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate

Toxicological profile of Umbilical Cord Blood-derived small Extracellular Vesicles

“…On the other hand, multiple reports demonstrate EV's potential to induce the proliferation of dermal cells, namely fibroblasts [26][27][28] . These outcomes are in line with previous UCB-MNC-sEV effects observed in wound healing, both in in vitro and in vivo models 12,19 .…”

“…Finally, the concentrated conditioned medium underwent size exclusion chromatography and EV-enriched fractions were collected, concentrated, and stored at -80ºC until further use. A more detailed description of the EV purification process is published elsewhere (19).…”

“…In this study, EV isolated from human umbilical cord blood mononuclear cells (UCB-MNC) through a clinically transferable process were tested for their toxicity in vitro and in vivo. As described in a recent paper, the optimized methodology combines ultrafiltration and size exclusion chromatography (UF/SEC), yielding a small EV (sEV)-enriched product, with particle sizes ranging from 50-200 nm 19 . In comparison with ultracentrifugation, UF/SEC significantly reduces production time and improves process standardization 20,21 , while maintaining sEV's bioactivity 19 .…”

“…However, we cannot predict if these sEV induce metabolic or cell viability alterations, bring inflammatory and immune responses or hematologic variations. UCB-MNC-sEV preparations consist of 80% sEV and 20% larger vesicles, as well as proteins and lipids 19 . So far, intravenous injection of similar EV showed no signs of toxicity 18 .…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Potential Therapeutic Strategy for Acute Kidney Injury

“…Ensuring the quality of EVs is an important problem, and producers are supposed to confirm that the EVs are derived from cellular matrix, rather than being components from cells damaged during cryopreservation and mechanical failure (130,131,135). It is advisable to optimize and confirm the preservation conditions to maintain the efficacy of the EVs after defrosting (128). Importantly, the follow-up time in previous studies was only between 1 day and 2 weeks, which is inadequate for evaluating the long-term outcome and prognosis (114), and more clinical trials with long-term follow-up are therefore needed to provide more robust evidence.…”