Kaoru Uesugi

Measurement system for biomechanical properties of cell sheet

In this study, we present a new fixture (self-attachable fixture) and tensile test system for measuring mechanical properties of cell sheet. To evaluate strength of cell sheet, it is the most important to measure mechanical properties of tensile mode. However, there has been no study which measured the tensile mechanical properties of cell sheet, since it has been difficult to attach a cell sheet in the tensile test system owing to the structure of the conventional fixture, and there has been no tensile test system which had a measurement range that covered the tension force range of the cell sheets. Therefore, we have addressed these problems by developing a self-attachable fixture and a tensile test system. By using developed system, we measured mechanical properties (tension, stress and initial stiffness) C2C12 of cell sheet cultured in different recipe of culture medium. The initial stiffness of cell sheet cultured in culture medium without FBS had a tendency to become stiffer. This indicates that our new fixture and test system are applicable for evaluating mechanical properties of cell sheets.

Development of cell-sheet handling tool for measurement of cell sheet adhesion force

We have demonstrated hybrid micro mechanical systems (HMMS) using muscle cells. When constructing cells to HMMS, there are some forces between adhesive interface and cells, dur to contraction of muscle cells must be considered to design. When these forces are larger than adhesion force of cells, cells detach from substrates and system is not functional. Measuring the adhesion force of cells could prevent from the breakage of the system. In this paper, we developed cell adhesion force measurement system “90 degree detachment examination” which defined by “International Organization for Standardization (ISO)”. To attempt 90 degree detachment examination, we noticed that to use cell sheet which cultured confluent on two-dimensional surface. And to use cell sheet, we develop four types of cell sheet handling tools. As a result, we measured maximum adhesion strength of cells about 10 N/mm.

Analytical Model and Experimental Evaluation of the Micro-Scale Thermal Property Sensor for Single-Sided Measurement

We report a new analytical model of the MEMS-based thermal property sensor for samples which are difficult to handle and susceptible to damage by thermal stimulus, such as living cells. Many sensor designs had been reported for thermal property measurements, but only a few of them have considered the analytical model of the single-sided measurement in which a measurement sample is placed on the sensor substrate. Even in the few designs that have considered the analytical model, their applicable limits are restricted to more than 1 mm length in practical situations. Our new model considers both the sample and the sensor substrate thermal properties and is applicable to a sensor length less than 1 µm. In order to minimize the influence of the heat stimulus to the sample, the model formulates the required heat dissipating time for different sensor geometries. We propose fast and precise detection circuit architecture to realize our model, and we discuss the sensor performance for a number of different designs.

A Closed System for Pico-Liter Order Substance Transport from a Giant Liposome to a Cell

In single cell analysis, transport of foreign substances into a cell is an important technique. In particular, for accurate analysis, a method to transport a small amount (pico-liter order) of substance into the cell without leakage while retaining the cell shape is essential. Because the fusion of the cell and the giant liposome is a closed system to the outside, it may be possible to transport a precise, small amount of substances into the cell. Additionally, there is no possibility that a leaked substance would affect other systems. To develop the liposome-cell transportation system, knowledge about the behavior of substances in the liposome and the cell is important. However, only a few studies have observed the substance transport between a liposome and a cell. Here, we report observation of small amount of substance transport into a single C2C12 cell by using a giant liposome. Substance transport occurred by electrofusion between the cell and the giant liposome containing the substance, which is a closed system. First, to observe the electrofusion and substance transport from the moment of voltage application, we fabricated a microfluidic device equipped with electrodes. We introduced suspensions of cells and liposomes into the microfluidic device and applied alternating current (AC) and direct current (DC) voltages for electrofusion. We observed a small amount (22.4 ± 0.1%, 10.3 ± 0.4% and 9.1 ± 0.1%) of fluorescent substance (Calcein) contained in the liposomes was transported into the cell without leakage outside the cell, and we obtained the diffusion coefficient of Calcein in the cell as 137 ± 18 μm2/s. We anticipate that this system and the knowledge acquired will contribute to future realization of more accurate single cell analysis in a wide range of fields.

Effect of mechanical stimulation on neurite outgrowth of dorsal root ganglion neurons toward integrative mechanobiologic nerve bridge

Many methods to promote outgrowth of neurons have been studied, including electrical stimulation, micropatterned substrate, neurotrophic factors, and optical stimulation. Neuron is known to have mechanotransducer proteins. Calcium influx through mechanosensitive ion channels take part in the neuronal response to mechanical stimuli. In this report, we demonstrate the effect of mechanical stretching to dorsal root ganglion neuron (DRG neuron). By stretching DRG neurons under some conditions, we succeeded enhancing the neurite outgrowth by forty-eight percent compared to non-stretched DRG neurons. Stimulation frequencies between 0.25 Hz and 1 Hz have less effect on enhancing the neurite outgrowth, but stimulation time has effect on it. The more time DRG neurons were stretched, the more DRG neurons were peeled off. We need to find the optimum stimulating condition. These results indicate that mechanical stimulation has a potential to be applied a functional artificial nerve bridge that promotes the outgrowth of neurons.

Evaluation system for mechanobiology of three-dimensional tissue multilayered in vitro

This paper reports the mechanical properties of 3D layered tissue were measured by using a special fixture (micro vacuum chuck) and a measurement system. In this study, the mechanical properties of 3D layered tissue were measured by tensile test. Since difficulty of fixing the 3D layered tissue to tensile test system, the micro vacuum chuck which could fix the 3D layered tissue to tensile test system without damage was developed. Additionally, because there has been no system which can measure tensile force of 3D layered tissue, special tensile system was also developed. By using micro vacuum chuck and developed system, the tensile force of 3D layered tissue was measured and the maximum force was about about 907μN (Strain = 1.35).

Behavior of a single muscle cell bioactuator on a biocompatible fiber string structure

In this paper, we proposed a micromachine that driven by a single muscle cell bioactuator. Dorsal vessel cells (DVCs) were used as a bioactuator because of their robustness. We adopted flagella movement as motion of the micromachine. In order to confirm the motion of the micromachine by DVCs, we assumed a physical model and estimated the maximum displacement of the micromachine. DVCs adhesion to the structure was also investigated. As a result, we confirmed DVCs adhesion and compared the behavior with control. DVCs on a structure contracted at a speed of 86% of DVCs on a dish. This result will lead the micromachine driven by a single muscle cell bioactuator.