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.
Highlights
The field of regenerative medicine has produced significant and innovative discoveries related to the use of stem cells to heal human tissue (Pati et al, 2015)
To characterize the purified exosomes derived from human adipose-derived stem cells (hADSCs) via UC and UF techniques, Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM), and western blot analysis were used
After confirming that the diameters of the particles are within the expected range for exosomes (50–150 nm), we further verified that these particles correspond to exosomes by using TEM imaging, a well-accepted technique for nanoparticle identification
Summary
The field of regenerative medicine has produced significant and innovative discoveries related to the use of stem cells to heal human tissue (Pati et al, 2015). The primary mechanism of action by which these cells are thought to improve tissue healing is via the release of secreted factors (e.g., growth factors and cytokines) that function in a paracrine manner (Caplan and Dennis, 2006). Exosomes are small (150 nm) membranebound particles that carry proteins, microRNAs (miRNAs), and mRNAs (Hodges et al, 2017; Murphy et al, 2018). These nanoparticles are endocytosed by recipient cells, and are key players in cell-to-cell communication (Thery, 2011). EVs produced by MSCs improve cell survival in the heart and kidney following ischemia-reperfusion injury (Arslan et al, 2013; Chen et al, 2013) as well as in the fetal brain after hypoxia-ischemia (Ophelders et al, 2016) and EVs can support peripheral nerve regeneration (Ching and Kingham, 2015)
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