Human Biofluid Image Inventory: Electron Microscopy Analysis of Circulating Particles from cf-mtDNA-enriched Biofluids

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 vesicular structures across human biofluids, 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 participants (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 to confirm enrichment. The resulting catalog of the most common circulating particles in plasma, serum, and saliva show that circulating double-membrane extracellular particles 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 and largely qualitative results do not directly demonstrate but are consistent with the concept of mitochondria transfer and/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.