Transferring isolated mitochondria into tissue culture cells

Abstract

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.

Modifying the Mitochondrial Genome

“…This small orifice leads to frequent clogging during delivery of large cargo, such as mitochondria. To work around this problem, a 6 micron needle was used to inject mitochondria into rodent eggs that are larger and more mechanically forgiving, perhaps because of a thick zona pellucida, than somatic cells (Yang and Koob, 2012). Subsequently, a 20 micron needle removed a portion of the cytoplasm containing the injected mitochondria, and these mitocytoplasts were fused to mouse ρ0 somatic cells using a viral-based membrane fusion technique (Yang and Koob, 2012).…”

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells

“…Successful approaches include cytoplasmic fusion between enucleated mitochondria donor cells and mtDNA eliminated ρ0 cells to generate transmitochondrial cybrid cell lines (Moraes et al, 2001). Also, direct microinjection of isolated mitochondria into somatic cells or oocytes (King and Attardi, 1988; Yang and Koob, 2012) and the transfer of isolated mitochondria, or mitochondrial transfer between cells, in vivo or in co-culture have been reported (Caicedo et al, 2015; Islam et al, 2012; Liu et al, 2014; Spees et al, 2006). However, microinjection is inefficient and it remains unclear whether tunneling nanotube transfer or the ‘spontaneous’ uptake of isolated mitochondria are general phenomena or condition/cell type specific mitochondrial transfer mechanisms.…”

Prospects for therapeutic mitochondrial transplantation

“…Direct microinjection of isolated mitochondria into cultured cells was thought to be impractical based on the size of mitochondria compared to the gauge of injection needles (Yang & Koob 2012), despite that previous studies used needles with tip diameter of 1 µm or less (King & Attardi 1988). To counteract this problem, mitochondria were first injected into oocytes to allow larger diameter needles to be used and then a mitocytoplast containing the cell membrane, cytoplasm, and the previously injected mitochondria was taken from the oocyte (Yang & Koob 2012).…”