Visualizing Intercellular Mitochondrial Transfer

Abstract

Intercellular mitochondrial transfer is an emerging therapeutic strategy for restoring cellular function in diseases associated with damaged mitochondrial DNA (mtDNA). However, elucidating the precise dynamics of this process requires robust and long-term tracking tools. Conventional chemical dyes suffer from phototoxicity and signal fading, whereas DsRed, a commonly used red fluorescent protein, forms toxic multimers that accumulate in lysosomes, leading to potential false-negative transfer analyses and a rapid loss of expression over time. To overcome these limitations, we evaluated TurboRFP equipped with a mitochondrial targeting signal (TurboRFPmt) as a superior tracking alternative. Compared to DsRed, TurboRFPmt demonstrated precise mitochondrial colocalization, significantly reduced nonspecific aggregation, higher brightness, and long-term stability without impairing cell proliferation. To validate its efficacy, we employed human amniotic epithelial cells (hAECs)—selected for their stemness and high genomic stability—as mitochondrial donors, and H₂O₂-treated HEK293T cells or HLE r 0 cells as mtDNA damage models. Using 3D confocal microscopy and 24-hour time-lapse imaging, we successfully captured the active transfer and stable cytoplasmic integration of TurboRFP-labeled healthy mitochondria from hAECs into damaged recipient cells. These findings establish TurboRFPmt as a highly reliable and essential tool for advancing the study of intercellular mitochondrial transfer.