A dual-sided Transwell co-culture system reveals mitochondrial cargo within tunneling nanotubes
Year: 2026
Type: article
Abstract: 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.
Source: Frontiers in Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W7225832037