A dual-sided Transwell co-culture system reveals mitochondrial cargo within tunneling nanotubes

Introduction Tunneling nanotubes (TNTs) are membranous intercellular conduits capable of transferring cellular cargo, yet directly linking TNT architecture with cargo transport remains technically challenging in conventional co-culture settings. Methods Interacting cell populations were spatially separated on opposite surfaces of a porous Transwell membrane, allowing individual TNTs and their luminal contents to be resolved in three dimensions. Three-dimensional confocal imaging, complementary cytoskeletal labeling, serial-section transmission electron microscopy, mitochondrial immunolocalization, and species-specific mitochondrial genotyping were used to characterize transmembrane TNTs and their cargo. Results α-Tubulin-, F-actin-, and β-actin-positive TNTs were observed traversing membrane pores across multiple co-culture configurations, including those containing primary cells. Serial-section transmission electron microscopy showed that these pore-spanning TNT-like structures possessed non-empty lumens containing cytoplasm-like material. COXIV-positive mitochondrial signals were localized within β-actin-positive transmembrane TNTs, providing direct spatial evidence of mitochondrial cargo within these intercellular conduits. Species-specific genotyping of mitochondrial 12S and 16S rRNA regions further detected donor-derived mtDNA in recipient human cells following co-culture. Discussion These findings support a role for transmembrane TNTs in intercellular mitochondrial material transfer and demonstrate the value of spatially resolved co-culture for investigating TNT-associated cargo exchange.