Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells.

EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, -20 °C, and -80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing -80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at -80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors). Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)'s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.