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bioRxiv · 10.1101/2025.06.15.658690

Radiolabeling Isolated Mitochondria with Tc-99m: A First-in-Field Protocol and Early Feasibility Findings

Walker, M.·Miralles, F.·Prijoles, K. L.·Kazmi, J.·Hough, J.·Lewis, D. H.·Levitt, M.·Sancak, Y.
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Abstract

Mitochondrial transplantation is a promising but still experimental strategy for treating ischemic and metabolic disorders. A key barrier to its advancement is the lack of scalable, non-invasive methods for tracking transplanted extracellular mitochondria in vivo. Technetium-99m (Tc-99m) radiopharmaceuticals, widely used in SPECT imaging, may offer a clinically compatible solution. Cryopreserved mitochondria derived from HEK-293 cells were incubated with Tc-99m sestamibi, tetrofosmin, pertechnetate, or control solutions. After brief incubation and washing, mitochondrial pellets were analyzed for retained radioactivity. ATP content was measured to assess metabolic function, and electron microscopy was used to evaluate ultrastructural integrity. Tc-99m sestamibi and tetrofosmin showed labeling efficiencies of 2.74% and 2.68%, respectively. Pertechnetate demonstrated minimal uptake (0.34%). Radiolabeled mitochondria retained ATP production comparable to controls. Electron microscopy showed preserved double membranes and cristae. Controls confirmed assay specificity and viability. To our knowledge, this is the first report of radiolabeling isolated mitochondria with clinically approved Tc-99m agents. This platform supports the development of SPECT-compatible protocols for visualizing viable transplanted mitochondria in recipient tissues.

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Walker, M., Miralles, F., Prijoles, K. L., Kazmi, J., Hough, J., Lewis, D. H., Levitt, M., Sancak, Y.. 2025-06-19. Radiolabeling Isolated Mitochondria with Tc-99m: A First-in-Field Protocol and Early Feasibility Findings. https://doi.org/10.1101/2025.06.15.658690

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