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 up-scalable 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 up-scaling 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 skin regeneration in a mouse model of delayed wound healing.The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and up-scalability required for mass production of sEV to be used in a clinical setting.

Stem cell‐mediated angiogenesis in skin tissue engineering and wound healing

“…44 To date, several studies have demonstrated the pro-angiogenic and skin tissue regeneration potential of UCB-MSCs-derived exosomes both in vitro and in vivo. 87 This capacity may be due to a two-step process, that is, (I) a first transient and immediate-acting secreted cytokines and other soluble factors and (II) exosomedelivered nucleic acids (e.g., miRNAs and mRNAs) for a prolonged response. 88 In this regard, Montemurro et al assessed the proangiogenic and anti-inflammatory behaviour of UCB-MSCs in terms of their soluble secretomes and EVs.…”

MSCs-Derived miR-150-5p-Expressing Exosomes Promote Skin Wound Healing by Activating PI3K/AKT Pathway through PTEN

“…Previous studies have shown that miR-150-5p in small extracellular vesicles plays a role in promoting healing by increasing skin neovascularization and re-epithelialization at the tissue/cell level (15,20). To study the effect of miR-150-5p expressed in the MSCs-Exos on H2O2-treated HaCaT cells, miR-150-5p expression was detected in HaCaT cells treated with different concentrations of H2O2.…”

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

“…Several studies have focused on improving MSC-EVs in a variety of areas ( 128 ) ( Table 1 ). These studies have mainly involved improving the technology for the isolation of EVs, investigating potential administration routes, the use of pulsed focused ultrasound (pFUS), preconditioning EVs, and inducing the overexpression of EVs by lentiviral vector transduction.…”

“…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.…”