Satoshi Migita

Development of sensor cells using NF-κB pathway activation for detection of nanoparticle-induced inflammation.

The increasing use of nanomaterials in consumer and industrial products has aroused concerns regarding their fate in biological systems. An effective detection method to evaluate the safety of bio-nanomaterials is therefore very important. Titanium dioxide (TiO2), which is manufactured worldwide in large quantities for use in a wide range of applications, including pigment and cosmetic manufacturing, was once thought to be an inert material, but recently, more and more studies have indicated that TiO2 nanoparticles (TiO2 NPs) can cause inflammation and be harmful to humans by causing lung and brain problems. In order to evaluate the safety of TiO2 NPs for the environment and for humans, sensor cells for inflammation detection were developed, and these were transfected with the Toll-like receptor 4 (TLR4) gene and Nuclear Factor Kappa B (NF-κB) reporter gene. NF-κB as a primary cause of inflammation has received a lot of attention, and it can be activated by a wide variety of external stimuli. Our data show that TiO2 NPs-induced inflammation can be detected by our sensor cells through NF-κB pathway activation. This may lead to our sensor cells being used for bio-nanomaterial safety evaluation.

Cell cycle and size sorting of mammalian cells using a microfluidic device

Microfluidic devices can sort viable mammalian cells by size. In this study, we investigated size-based sorting of cells using flow splitting microfluidic devices based on hydrodynamic filtration for noninvasive cell cycle synchronization. Two different types of mammalian cell lines, HepG2 (human hepatocellular liver carcinoma cell line) and NIH/3T3 (mouse embryonic fibroblast cell line) were sorted by microfluidic device and its DNA contents were analyzed. Our results showed that a microfluidic device can synchronize the cell cycle after size separation. The damage-free separation of living cells in different phases of the cell cycle represents a potentially promising technology for the investigation of gene transfection and gene expression.

Discerning Data Analysis Methods to Clarify Agonistic/Antagonistic Actions on the Ion Flux Assay of Ligand-Gated Ionotropic Glutamate Receptor on Engineered Post-Synapse Model Cells

Cell-based experiments provide the efficacy of chemicals through the biological function. The authors have described post-synapse model cell-based assay based on qualified analysis for neural drug discoveries. However, in general, cell-based assays often include data fluctuation. This may be a barrier preventing the performance for various practical purposes. In this study, we tried discerning data analysis for clarify the chemical action to the ionotoropic glutamate receptor (GluR), whereby an ion-flux assay of post-synapse model cells is performed and are analyzed based on a chemometrics approach. The dynamic behavior of the GluR of post-synapse model cell was assayed with multivariate data analysis methods namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). By using HCA, we can identify and remove the non-responding samples. By using PCA, the effect of chemicals on the dynamic behavior of ion flux through GluR can be recognized clearly; as either agonist or antagonist. As shown in the results, the GluR-based assay by post-synapse model cell with data analysis methods provide a sodium influx profile which is derived by an agonists or antagonists application. By employing the data analysis methods, PCA and HCA, it is possible to develop a smart cellular biosensing system for qualified analysis.

In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

Abstract Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ∼67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

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., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high‐throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell‐based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum‐starved conditions and we then investigated the cadmium chloride‐induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell‐based biosensors with microdevices, such as a single cell array. Biotechnol. Bioeng. 2010;107: 561–565.

Transfection efficiency for size-separated cells synchronized in cell cycle by microfluidic device

Non-viral system generally demonstrates less efficacious in transgene delivery than viral system; however it represents a safer alternative to viral system. In this study, transfection efficiency for human hepatocellular liver carcinoma cells synchronized in cell cycle at G0/G1 phase, which was sorted in size with a microfluidic device based on hydrodynamic filtration, was investigated by using a reverse transfection method. The synchronized cells were recovered at the yield of 80% from the micro-channel, and green fluorescent protein gene encoding plasmid mixed with lipofectoamine was transfected. The transfection efficiency of the cells at G0/G1 phase was 1.8 times higher than non-synchronized cells. The manipulation of cell cycle status could increase transfection efficiency in non-viral system, indicating size-based cell cycle synchronization is a powerful tool as a noninvasive method for bioscience and biotechnology.

Molecular Commonality Sensing of Phosphoric Anhydride Substances Using an Ion-Sensitive Field-Effect Transistor Covered with an Artificial Enzyme Membrane

A new type of biosensor has been developed using an artificial enzyme membrane that recognizes and hydrolyzes phosphoric anhydride bonds selectively. The artificial enzyme (artificial P–P hydrolase) membrane was designed and synthesized by means of our original procedure, and mounted on a T2O5-gate ion-sensitive field-effect transistor (ISFET). H2PO4- ions are produced through the catalytic reaction in the artificial P–P hydrolase membrane, and accumulate in the membrane on the ISFET. The accumulated H2PO4- ions cause a shift in the static ISFET output as the sensor response. A key advantage of the artificial P–P hydrolase is its specific structure selectivity for the phosphoric anhydride bonds, which is one of the dominant and characteristic structures in biological energy substances. The molecular commonality detection of the phosphoric anhydride bonds is a novel tactic for biosurveillance and cellular viability assays.

Quantum dots induce heat shock-related cytotoxicity at intracellular environment

Quantum dots (QDs) are semiconductor nanocrystals with unique optical properties. Different proteins or polymers are commonly bound to their surfaces to improve biocompatibility. However, such surface modifications may not provide sufficient protection from cytotoxicity due to photodegradation and oxidative degradation. In this study, the cytotoxic effects of QDs, CdTe, and CdSe/ZnS were investigated using cadmium-resistant cells. CdTe QDs significantly reduced cell viability, whereas, CdSe/ZnS treatment did not markedly decrease the cell number. CdTe QDs were cytotoxic in cadmium-resistant cells suggesting that internalized QDs degraded and cadmium ions contributed to the cytotoxic effects. CdTe QDs were consistently more cytotoxic than CdSe/ZnS QDs, but both QDs as well as cadmium ions activated heat shock protein 70B′ promoter. QDs themselves are likely to contribute to HSP70B′ promoter activation in cadmium-resistant cells, because CdSe/ZnS QDs do not release sufficient cadmium to activate this promoter.

Post-synapse model cell for synaptic glutamate receptor (GluR)-based biosensing: strategy and engineering to maximize ligand-gated ion-flux achieving high signal-to-noise ratio.

Cell-based biosensing is a “smart” way to obtain efficacy-information on the effect of applied chemical on cellular biological cascade. We have proposed an engineered post-synapse model cell-based biosensors to investigate the effects of chemicals on ionotropic glutamate receptor (GluR), which is a focus of attention as a molecular target for clinical neural drug discovery. The engineered model cell has several advantages over native cells, including improved ease of handling and better reproducibility in the application of cell-based biosensors. However, in general, cell-based biosensors often have low signal-to-noise (S/N) ratios due to the low level of cellular responses. In order to obtain a higher S/N ratio in model cells, we have attempted to design a tactic model cell with elevated cellular response. We have revealed that the increase GluR expression level is not directly connected to the amplification of cellular responses because the saturation of surface expression of GluR, leading to a limit on the total ion influx. Furthermore, coexpression of GluR with a voltage-gated potassium channel increased Ca2+ ion influx beyond levels obtained with saturating amounts of GluR alone. The construction of model cells based on strategy of amplifying ion flux per individual receptors can be used to perform smart cell-based biosensing with an improved S/N ratio.

Engineered synapse model cell: genetic construction and chemical evaluation for reproducible high-throughput analysis.

Bioassay models of neural functions must lend themselves to high-throughput analysis in neural drug discovery. However, smart analysis methods for these functions have not yet been fully established. Here, we describe the development of a synapse model for cell-based biosensing. The engineered synapse model cell expresses ionotropic glutamate receptor on its surface, like the neural postsynaptic membrane. The advantages of the model cell are the ease of handling and reproducibility as compared with the cultured neural cell, and it can be employed to evaluate receptor function through ion flux analysis. The agonist-induced sodium influx was monitored as an agonist concentration-dependent increase in the observed fluorescence signal. Furthermore, we found that our model cell enables the correction of uneven cellular signal levels using a reporter system. Our engineered synapse model cell can be employed as a powerful tool for the screening of lead substances in pharmaceutical high-throughput analysis.