Mitochondrial transfer between cells: Methodological constraints in cell culture and animal models

Interest in the recently discovered phenomenon of mitochondrial transfer between mammalian cells has gained momentum since it was first described in cell culture systems more than a decade ago. Mitochondria-targeting fluorescent dyes have been repurposed and are now widely used in these studies and in acute disease models, sometimes without due consideration of their limitations, while vectors containing mitochondrially-imported fluorescent proteins have complemented the use of mitochondria-targeting dyes. Genetic approaches that use mitochondrial DNA polymorphisms have also been used in some in vitro studies and in tumor models and are particularly useful where mtDNA is damaged or deleted. These approaches can also be used to study the long-term consequences of mitochondrial transfer such as in bone marrow and organ transplantation and in tumour biology where inherent mitochondrial damage is often a key feature. As research on intercellular mitochondrial transfer moves from cell culture into animal models and human diseases it will be important to understand the limitations of the various techniques in order to apply appropriate methodologies to address physiological and pathophysiological conditions. •MitoTracker dyes can leak and can be toxic to cells under certain conditions.•Fluorescent proteins do not leak from cells.•Mitochondrial DNA polymorphisms are useful tools in short and long-term studies.•Location of acquired mitochondria must be validated by z stacks and deconvolution.•Restoration of mitochondrial function validates the importance of transfer.