BACKGROUND Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously. METHODS The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface. RESULTS The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay. CONCLUSION Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes. LEVEL OF EVIDENCE Therapeutic care management, level II.

Background Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold‐stored platelets (CSPs) and RTPs after 5‐day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days. Study Design and Methods This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial‐associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics. Results CSP preserved mitochondria‐associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro‐apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs. Conclusion Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well‐documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.

BACKGROUND Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration. METHODS The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine. RESULTS The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak. CONCLUSION Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential. LEVEL OF EVIDENCE Basic or Foundational Research.