Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury

Abstract

Cellular therapies have tremendous potential for the successful treatment of major extremity wounds in the combat setting, however, the challenges associated with transplanting stem cells in the prolonged field care (PFC) environment are a critical barrier to progress in treating such injuries. These challenges include not only production and storage but also transport and handling issues. Our goal is to develop a new strategy utilizing extracellular vesicles (EVs) secreted by stem cells that can resolve many of these issues and prevent ischemic tissue injury. While EVs can be preserved by freezing or lyophilization, both processes result in decrease in their bioactivity. Here, we describe optimized procedures for EVs production, isolation, and lyophilization from primary human adipose-derived stem cells (hADSCs). We compared two isolation approaches that were ultrafiltration (UF) using a tangential fluid filtration (TFF) system and differential ultracentrifugation (UC). We also optimized EVs lyophilization in conjunction with trehalose and polyvinylpyrrolidone 40 (PVP40) as lyoprotectants. Bioactivity of EVs was assessed based on reversal of hypoxia-induced muscle cell injury. To this end, primary human myoblasts were subjected to hypoxic conditions for 6 h, and then treated with hADSC-derived EVs at a concentration of 50 µg/mL. Subsequently, muscle cell viability and toxicity were evaluated using MTS and LDH assays, respectively. Overall, nanoparticle tracking data indicated that UF/TFF yields threefold more particles than UC. Lyophilization of EVs resulted in a significantly reduced number of particles, which could be attenuated by adding lyoprotections to the freeze-drying solution. Furthermore, EVs isolated by UF/TFF and freeze-dried in the presence of trehalose significantly increased viability (P < 0.0193). Taken together, our findings suggest that the isolation and preservation methods presented in this study may enhance therapeutic applications of EVs.

COVID‐19 therapy with mesenchymal stromal cells (MSC) and convalescent plasma must consider exosome involvement: Do the exosomes in convalescent plasma antagonize the weak immune antibodies?

“…The safety of exosomes in humans is emphasized by the fact that there have been billions in blood plasma transfusions containing trillions of exosomes over many decades in diverse patients without problems, whereas MSC as cells are difficult to standardize or predict actions of given lots in vivo in patients. On a practical level MSC exos can be freeze‐thawed at least once without toxic cryo‐preservatives, with no lost activity for months to years, and it has been reported that they can be freeze‐dried (El Baradie et al., 2020), for superior storage, allowing on site administration without refrigeration. This can result in uncomplicated use on hospital wards or in poorer countries with low technology medical facilities.…”

The cyclophilin inhibitor NIM-811 increases muscle cell survival with hypoxia in vitro and improves gait performance following ischemia–reperfusion in vivo

“…Primary human skeletal myoblasts purchased from ThermoFisher Scientific (Catalog # A11440) were cultured as monolayers in Dulbecco's Modified Eagle Medium (DMEM/High Glucose), supplemented with 5% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (Pen-Strep) as we described previously 41 . The medium was changed after 24 h to discard non-adherent cells.…”

“…A stock solution of N-methyl-4-isoleucine cyclosporine NIM-811 (NIM-811; MedChemExpress, NJ, USA) was prepared by dissolving in DMSO at a concentration of 1 mM. As we described previously 41 , different volumes of the stock solution were added to myoblast cultures during the last 20 min of the hypoxia-period to obtain (0–20 µM) final concentrations of NIM-811 in the culture medium (phenol red-free DMEM supplemented with 1% FBS). At the end of the hypoxia period the myoblast cultures were transferred to a normoxic incubator for an additional 2 h.…”

“…After the ischemic human myoblasts were treated with NIM-811 as detailed above, the MTS and LDH assays were performed as we showed previously to assess the tissue damage after I/R 41 . In brief, in each well the ischemic myoblasts after the removal of the culture medium, were mixed with 100 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS, Promega Corporation, FL, USA).…”

Extracellular Vesicles for the Treatment of Radiation Injuries

“…More GMP suitable cryopreservation reagents need to be developed for EV preservation. On other hand freeze-dried EVs can be a greater source for long-term storage and therapeutic applications ( El Baradie et al, 2020 ).…”