Stability of Stored Melanoma Microvesicles Determined by Optical Imaging and Macrophage Activation Potential

Extracellular vesicles show great potential for use in diagnostics, due to their presence in biological fluids such as urine and blood, and their implication in diseases such as cancer and neurodegenerative disorders. However, the effect of storage on their properties remains unknown. In this report, we investigate the time-varying properties of murine melanoma microvesicles (MVs) after storage at different temperatures. The viability of the vesicles was investigated through fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), particle sizing measurements, and Raman spectroscopy.  Applying FLIM to evaluate stored MVs is a novel method to discover differences in NAD(P)H content that provide evidence for the degradation or integrity of the MVs after storage. Results demonstrate that storage may affect the integrity of microvesicles stored at  20ºC more than those stored at 4ºC, and that our centrifugation protocols likely left the sample with residual melanin that deters further investigation with the melanoma cell line to understand how the degradation of MVs affects living cells.