Generation of transmitochondrial cybrids using a microfluidic device.

Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device.

Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.

Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.

A Microfluidic Device for Modulation of Organellar Heterogeneity in Live Single Cells.

The quantitatively controlled organellar transfer between living single cells provides a unique experimental platform to analyze the contribution of organellar heterogeneity on cellular phenotypes. We previously developed a microfluidic device which can perform quantitatively controlled mitochondrial transfer between live single cells by promoting strictured cytoplasmic connections between live single cells, but its application to other organelles is unclear. In this study, we investigated the quantitative properties of peroxisome transfer in our microfluidic device.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed.

Quantitative control of mitochondria transfer between live single cells using a microfluidic device.

Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because single mitochondrion transfer to a mtDNA-less (ρ) cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.

Cryopreservation of adhered mammalian cells on a microfluidic device: Toward ready-to-use cell-based experimental platforms.

In this paper, we describe cryopreservation of mammalian cells in the adhered state on a microfluidic device (microdevice) for the first time. HeLa, NIH3T3, MCF-7, and PC12 cells were cultured on a microdevice in which a commercial polystyrene dish surface was used as the cell adhesion surface. Without cell-detaching treatment, the microdevice was stored in a freezer at -80°C.

The significance of membrane fluidity of feeder cell-derived substrates for maintenance of iPS cell stemness.

The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs.

Effects of ROCK inhibitor Y-27632 on cell fusion through a microslit.

We previously reported a direct cytoplasmic transfer method using a microfluidic device, in which cell fusion was induced through a microslit (slit-through-fusion) by the Sendai virus envelope (HVJ-E) to prevent nuclear mixing. However, the method was impractical due to low efficiency of slit-through-fusion formation and insufficient prevention of nuclear mixing. The purpose of this study was to establish an efficient method for inducing slit-through-fusion without nuclear mixing.

Microfluidic perfusion cell culture system confined in 35 mm culture dish for standard biological laboratories.

An extremely simple, self-standing microfluidic cell culture system is reported. The whole system is confined in a 35 mm culture dish, and requires only a standard CO2 incubator. The culture medium is perfused by gravity.

Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.

Hippo pathway regulation by cell morphology and stress fibers.

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor.

Modulating F-actin organization induces organ growth by affecting the Hippo pathway.

The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood.

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e.

[Development of novel culture system using nano-biotechnology].

We have developed two cell culture systems for use in pharmaceutical research using nano-biotechnology. First, we developed a double layered co-culture system using cell sheet technology, and showed that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various cytochrome P450 (CYP) genes were significantly increased compared to monolayer cultured HepG2 cells. In the layered HepG2 co-culture, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment.

DNA damage sensible engineered promoter for cellular biosensing of cytotoxicity.

We have established a cytotoxic sensor cell line by transfecting HepG2 cells with a luciferase protein plasmid derived from the heat shock protein 70B' (HSP70B') promoter, which is induced by cytotoxic reagents. HSP70B genes are up-regulated by a wide-range of cytotoxic stimulators, in particular, those that denature proteins. However, the HSP70B genes do not respond to DNA damage.

Formation process and fate of the nuclear chain after injury in regenerated myofiber.

Although it is well known that regenerated myofibers contain nuclear chains (arrayed nuclear clusters), details of its process of formation and fate are still remained unclear. In the present study, we isolated single myofibers from injured ICR mouse tibialis anterior muscles by the alkali maceration-based method, and carried out histological observation and bromodeoxyuridine (BrdU) pulse-chase analysis on the nuclear chains. The nuclear chains were formed after injury and remained stable for at least 6 months after injury.

Live cells-based cytotoxic sensorchip fabricated in a microfluidic system.

We report here a live cells-based sensorchip fabricated in microfluidic channels in which several laminar flows were achieved. In addition, we established a cytotoxic sensor cell line, which was transfected with a green fluorescence protein (GFP) plasmid derived from the heat shock protein 70B' (HSP70B') promoter, which is induced by cytotoxic reagents. The fluorescence in the sensor cells increased in a CdCl(2) dose-dependent manner in the microfluidic channels.

Highly sensitive detection of cytotoxicity using a modified HSP70B' promoter.

We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B' gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B' promoter.

Rapid and highly sensitive detection of cadmium chloride induced cytotoxicity using the HSP70B' promoter in live cells.

One unique to detect cytotoxicity is to utilize reporter gene assays for promoters that respond to stress-induced effects. In the present study, we discovered that the DNA sequence from nt -287 to +110 of the heat shock protein 70B' (HSP70B') gene could be used as a functional promoter to detect cytotoxicity of cadmium chloride. We thus detected cytotoxicity induced by cadmium chloride with the luciferase assay using this functional HSP70B' promoter, as well as the cell viability test based on the quantification of intracellular ATP.