Electron microscopy visualization of cell-free mitochondrial DNA-containing extracellular vesicles in human plasma, serum, and saliva

Human biofluids contain cell–free mitochondrial DNA (cf–mtDNA) and extracellular mitochondria (ex–Mito), creating the challenge of defining their origins, destinations, mechanisms of regulation, and purposes. To expand our understanding of cf–mtDNA biology, we present a descriptive electron microscopy analysis of circulating particles from cf–mtDNA–enriched plasma (citrate, heparin, and EDTA), serum (red and gold top), and saliva collected from ten healthy people (5 females, 5 males, mean age 44.9 years). Ex–mito and extracellular vesicles (EVs) were isolated by centrifugation followed by size–exclusion chromatography, imaged by transmission electron microscopy, and morphometrically analyzed. In parallel, cf–mtDNA was quantified in each biofluid. The resulting catalog of the most common circulating particles in plasma, serum, and saliva show that circulating double–membrane extracellular particles — consistent with mitochondrial ultrastructure — are present across human biofluids, along with EVs and other particle types. Combining imaging with cf–mtDNA quantification, we show that individuals with higher plasma cf–mtDNA concentrations tend to contain more double-membrane, ex–Mito–like particles. These preliminary results challenge the notion that, under normal conditions, the majority of cf–mtDNA exists as naked and potentially pro–inflammatory forms. Instead, these results are consistent with the concept of mitochondria transfer or signaling between cells and tissues. The image inventory provided here expands our knowledge of cell-free mitochondrial biology and provides a resource to inform biofluid selection and technical considerations in future studies quantifying ex–Mito and cf–mtDNA.