Comparative Assessment of Methods for Isolation and Imaging of Extracellular Vesicles

Extracellular vesicles (EVs) from defined cell sources are emerging as potential vehicles with therapeutic efficacy. The EV and non-EV secretome of individual cell populations may also be used as a measure of cell identity or state. EV attributes and composition may therefore be useful in defining the "fit-for-purpose" quality of cell populations for drug testing, biological models, and cell therapy products. As such, methods for cost-effective isolation and rigorous characterization of EVs are needed. This work aims to evaluate current methods and establish effective and potentially improved techniques to address these needs. First, ultracentrifugation, ultrafiltration, and precipitation methods were evaluated as means for EV isolation from culture-expanded human mesenchymal stromal cell (hMSC) populations. Second, traditional formvar and updated lacey carbon transmission electron microscopy (TEM) grid preparations were compared. Third, electron energy loss spectroscopy (EELS) was evaluated for EV elemental content characterization. Fourth, the efficacy of dynamic light scattering (DLS) was evaluated as a tool for characterizing variation in EV size distribution between hMSC populations. Finally, DLS measurements were compared to direct TEM size measurements using EV populations isolated from 28 culture-expanded hMSC populations derived from 5 human donors. Ultracentrifugation was most effective in providing higher EV purity and yield per visual TEM analysis. Lacey carbon TEM grids provided the most effective imaging for visual characterization of EV morphology and enabled durable archiving of samples for discontinuous assessment. EELS identified phosphorus in the lipid bilayer but was ineffective at identifying sulfur (protein) and nitrogen (protein, RNA, DNA) contents of EVs. DLS was generally consistent in characterizing EV population size distributions, but discrepancies and limitations were identified based on direct comparative TEM observation. This study demonstrated that ultracentrifugation, DLS size characterization, and confirmation with lacey carbon TEM grid preparation provided high quality samples and effective assessment of EV size distribution and morphology.