SUMMARY mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggest metabolite profiling can help assess the quality and function of mtDNA modified cells.

Summary Human mitochondria produce ATP and metabolites to support development and maintain cellular homeostasis. Mitochondria harbor multiple copies of a maternally-inherited, non-nuclear genome (mtDNA) that encodes for 13 subunit proteins of the respiratory chain. Mutations in mtDNA occur mainly in the 24 non-coding genes, with specific mutations implicated in early death, neuromuscular and neurodegenerative diseases, cancer, and diabetes. A significant barrier to new insights in mitochondrial biology and clinical applications for mtDNA disorders is our general inability to manipulate the mtDNA sequence. Microinjection, cytoplasmic fusion, nucleic acid import strategies, targeted endonucleases, and newer approaches that include the transfer of genomic DNA, somatic cell reprogramming, and a photothermal nanoblade, attempt to change the mtDNA sequence in target cells with varying efficiencies and limitations. Here, we discuss the current state of manipulating mammalian mtDNA and provide an outlook for mitochondrial reverse genetics, which could further enable mitochondrial research and therapies for mtDNA diseases.

Optoelectronic tweezers (OET), based on light-induced dielectrophoresis, has been shown as a versatile tool for parallel manipulation of micro-particles and cells (P. Y. Chiou, A. T. Ohta and M. C. Wu, Nature, 2005, 436, 370-372). However, the conventional OET device cannot operate in cell culture media or other high-conductivity physiological buffers due to the limited photoconductivity of amorphous silicon. In this paper, we report a new phototransistor-based OET (Ph-OET). Consisting of single-crystalline bipolar junction transistors, the Ph-OET has more than 500x higher photoconductivity than amorphous silicon. Efficient cell trapping of live HeLa and Jurkat cells in Phosphate Buffered Saline (PBS) and Dulbecco's Modified Eagle's Medium (DMEM) has been demonstrated using a digital light projector, with a cell transport speed of 33 microm/sec, indicating a force of 14.5 pN. Optical concentration of cells and real-time control of individually addressable cell arrays have also been realized. Precise control of separation between two cells has also been demonstrated. We envision a new platform for single cell studies using Ph-OET.

Flexible neural probes have been pursued previously to minimize the mechanical mismatch between soft neural tissues and implants and thereby improve long-term performance. However, difficulties with insertion of such probes deep into the brain severely restricts their utility. We describe a solution to this problem using gallium (Ga) in probe construction, taking advantage of the solid-to-liquid phase change of the metal at body temperature and probe shape deformation to provide temperature-dependent control of stiffness over 5 orders of magnitude. Probes in the stiff state were successfully inserted 2 cm-deep into agarose gel "brain phantoms" and into rat brains under cooled conditions where, upon Ga melting, they became ultra soft, flexible, and stretchable *

We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23,000 cells sec−1 with 90% purity in high-purity mode and at a throughput of 45,000 cells sec−1 with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μsec complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m sec−1) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter.