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.
Competing Interest Statement
R.M.S.C and J.S.C are inventors of the patent PCT/IB2017/000412 (Use of umbilical cord blood derived exosomes for tissue repair) and R.M.S.C., S.C.R and J.S.C. are inventors of the patent PCT/IB2019/058462 (Compositions comprising small extracellular vesicles derived from umbilical cord blood mononuclear cells with anti-inflammatory and immunomodulatory properties), currently explored by Exogenus Therapeutics, S.A. Financial interest is claimed by Exogenus Therapeutics, S.A., which holds a licence (PCT/IB2017/000412) and a patent related to this work (PCT/IB2019/058462), and J.S.C. in the capacity of a shareholder of Exogenus Therapeutics, S.A. The other authors declare no conflicts of interest.
Abbreviation list
- ACTB
- beta-actin
- CE
- cholesterol-fatty acid ester
- CM
- conditioned medium
- ECM
- extracellular matrix
- EV
- Extracellular Vesicles
- HUVEC
- human umbilical vein endotelial cells
- ISPs
- Ion Sphere Particles
- MNC
- mononuclear cells
- MSC
- Mesenchymal stem cells
- NDHF
- human dermal fibroblasts
- PC
- phosphatidylcholines
- PE
- phosphatidylethanolamine
- PS
- phosphatidylserines
- sEV
- small Extracellular Vesicles
- SPM
- sphingomyelin
- TAG
- triacylglyceride
- UC
- ultracentrifugation
- UCB
- umbilical cord blood
- UF/SEC
- ultrafiltration combined with size exclusion chromatography
- VLFAC
- very long fatty acid chains