Masaru Hakoda

Lethal effect of high-pressure carbon dioxide on a bacterial spore

Abstract To develop a novel and effective method for sterilization of bacterial spores, we examined the lethal effect of high-pressure CO 2 on spore cells of Bacillus megaterium at various pressures and temperatures and for various treatment times. The bactericidal effect was found to be enhanced with increasing temperature and treatment time. Interestingly, there was an optimum pressure in the subcritical region of CO 2 which gave the maximum inactivation effects. Under the appropriate conditions of 58 atm, 60°C and 30 h, the survival ratio of the bacterial spore could be reduced to about 10 −7 .

Development of a Method To Analyze Single Cell Activity by Using Dielectrophoretic Levitation

In cell fusion and genetic recombination, although the activity of single cells is extremely important, there is no method to analyze single cell activity. Development of a quick analyzing method for single cell activity is desired in various fields. Dielectrophoresis (DEP) refers to the force exerted on the induced dipole moment of an uncharged dielectric and/or conductive particle by a nonuniform electric field. By applying DEP, we obtained experimentally a relationship between the cell activity and the dielectric property, Re[K(ω)], and examined how to evaluate the single cell activity by measuring Re[K(ω)] of a single cell. A cone and plate electrode geometry was adapted in order to achieve the feedback‐controlled DEP levitation. The single cell is exposed to a nonuniform field induced by the cone and plate electrode, and a more polarizable cell is moved to the direction of the cone electrode by the DEP force. The cell settles in the position where the DEP force and gravity are balanced by controlling applied voltage. This settled position, measured on the center axis of the cone electrode, depended on the dielectric constant of the cell. From these results, the relationship between the specific growth rates in cell growth phase and the dielectric properties Re[K(ω)] was obtained. Furthermore, the effect on the cell activity of various stresses, such as concentration of carbon dioxide, temperature, etc., was examined.

Electro-ultrafiltration bioreactor for enzymatic reaction in reversed micelles

Electro-ultrafiltration (EUF) was applied to separate the Aerosol OT (AOT) reverse micelles containing lipase from isooctane, and the effect of electric field on the rejection of reverse micelles and the permeation flux was examined experimentally. It could be said from the experimental results that EUF was effective for separation of the reverse micelles containing the enzyme and that the motive forces for the negatively charged reverse micelles were electrophoretic and dielectrophoretic.

Relationship between Dielectric Characteristic by DEP Levitation and Differentiation Activity for Stem Cells

In our previous study, we discussed the possibility of differentiation activity measurement for rat mesenchymal stem cells (RMSC) by Dielectrophoretic (DEP) levitation. Consequently, it was found that the differentiation activity of the RMSC could be evaluated by DEP levitation without the differentiation induction. Thus, we discuss the possibility of differentiation activity evaluation by DEP levitation with cells other than the RMSC. Human mesenchymal stem cells (HMSC) and human adipose tissue-derived stem cells (ASC) were used as the sample cells. The dielectric characteristics (Re[K(ω)]) measurement, the Re[K(ω)] of both the HMSC and the ASC decreased with the increasing passage number. Moreover, to evaluate the differentiation activity of the HMSC and the ASC that had performed the osteoblast differentiation induction, the amount of Alkaline Phosphatase (ALP) was measured. Consequently, the ALP activity of both the HMSC and ASC decreased with increasing the passage number. Therefore, it was found that the differentiation activity of the HMSC and the ASC could be evaluated by measuring the Re[K(ω)] due to the relationship between the Re[K(ω)] and ALP activity.

Separation of viable and nonviable animal cell using dielectrophoretic filter

Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large‐scale separation because they cannot be performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large‐scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3‐2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed.

Separation characteristics of animal cells using a dielectrophoretic filter.

The separation characteristics of a wire–wire type dielectrophoretic (DEP) filter were evaluated using animal cells. The separation of cells with different activities was examined using a DEP filter. The specific growth rate of the cells in retention liquid was larger than that in permeation liquid. From the culture results of the separated cells, it becomes clear that the specific growth rate of the cells of the retention liquid was higher than that of the cells of the permeation liquid. Furthermore, as a result of separating cells two kinds of cell suspensions using the DEP filter, the difference between the retention ratios of the two groups of obtained cells was about 20% at maximum.

Development of dielectrophoresis separator with an insulating porous membrane using DC-Offset AC Electric Fields.

Our previous studies revealed that the dielectrophoresis method is effective for separating cells having different dielectric properties. The purpose of this study was to evaluate the separation characteristics of two kinds of cells by direct current (DC) voltage offset/alternating current (AC) voltage using an insulating porous membrane dielectrophoretic separator. The separation device gives dielectrophoretic (DEP) force and electrophoretic (EP) force to dispersed particles by applying the DC‐offset AC voltage. This device separates cells of different DEP properties by adopting a structure in which only the parallel plate electrodes and the insulating porous membrane are disposed in the flow path through which the cell‐suspension flows. The difference in the retention ratios of electrically homogeneous 4.5 μm or 20.0 μm diameter standard particles was a maximum of 82 points. Furthermore, the influences of the AC voltage or offset voltage on the retention ratios of mouse hybridoma 3‐2H3 cells and horse red blood cells (HRBC) were investigated. The difference in the retention ratio of the two kinds of cells was a maximum of 56 points. The separation efficiency of this device is expected to be improved by changing the device shape, number of pores, and pore placement.

Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field

The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.

Disruption of Microbial Cells by Flash Discharge of High Pressure Gas

To develop a novel sterilization method for heat-sensitive materials, we investigated the burst of microbial cells by rapid release of gas pressure under the various conditions. The wet cells of S. cerevisiae were well destroyed, when the organisms were saturated with CO2 gas at 40 °C and 40 atm and then the pressure was suddenly released. On the other hand, the dry cells were poorly disrupted even under the same experimental condition. In particular, the gas with low solubility in water could provide no effects on the survival ratio of the yeast. The survival ratio for the spores of B. megaterium QMB 1551 was also reduced, when the spores were applied to our system under the condition of 60 °C and 60 atm. From these findings, the mechanism of cell disruption is considered to be correlated with the gas absorption and desorption of the cells.

Development of Particle Packed Bed Type Chromatography Using Dielectrophoresis

In this study, we analyzed differences in cell species using dielectrophoresis (DEP) and propose a particle packed bed type chromatography device using DEP. Mouse-hybridoma 3-2H3 cells were used as the sample cells. The 3-2H3 cell suspension was fed pulse-wise to a carrier flow and the sample was measured by an absorption spectrometer. The analytical characteristic of chromatography using DEP was evaluated by the retention time difference. We examined the effects of the diameter of packed particles on the retention time difference. The retention time difference in the case of the packed particles of 100 μm diameter was larger than that of 500 μm, which suggests that packed particles of smaller diameter are suitable for analysis using DEP chromatography.