Ken-ichi Konno

Non-invasive Stiffness Detection Method for Living Cell Nucleus by Using Piezoelectric Micro Sensor

Cell nucleus is a body including various components and it has complicated and heterogeneous structure. Though most functions of nuclear domains have been well studied, mechanical properties of the nuclear domains has not been studied. In this study, the experimental system was developed to measure the stiffness of living cell nucleus in order to verify the inhomogeneity of the cell nucleus. The system is composed of two main devices: a sensor which can measure stiffness of micro cell nucleus and a rotatable device which can rotate nucleus horizontally. The devices are assembled on the stage of an inverted phase contrast microscope. Experimental studies have been carried out by using normal human osteoblast. The method has shown capability to detect difference of stiffness on cell nucleus. There is significant difference between nucleolus and other nuclear domains.

Novel Three-dimensional Micro Vibration Stage and Its Ability to Influence in Cellular Senescence

Although most cells are facing to mechanical stimuli, environment of cultured cells in an ordinary CO2 incubator may be inappropriate, because these cultured cells are normally free from mechanical stimuli. In this study, a novel three-dimensional micro vibration stage is developed to stimulate cultured cells mechanically. The simple two times twisted v-shaped steric structure of the stage makes possible to excite a culture dish three-dimensionally. By using the stage, the effect of vibration stimulation to adhesive cells was morphologically investigated. The experimental result using normal human osteoblast shows that the vibration stimulation decreases the projected area and increases the slenderness ratio of the cells. It suggests that the dynamic stimulation inhibits the morphological change according to progression of cellular senescence. A newly defined parameter, anti-aging effect, is proposed to describe the effect of the dynamic stimulation. It is found that the most effective vibration direction and frequency is unidirectional, horizontal and 10 Hz.

Three-Dimensional Micro Vibration Stage and Its Application to Cell Culture

One of the important roles of cytoskeleton is to maintain global mechanical strength of the cell. It is expected the effect of environmental mechanical stimulations appears in cell morphology. An environment of cultured cells in an ordinary incubator might be inappropriate, because those are normally free from mechanical stimulations such as fluid flow shear stress, tensile strain, compression and vibration. In this study, a three-dimensional micro vibration stage, to exert vibration stimulation non-invasively and three-dimensionally onto cultured cells, is developed. This vibration stage is assumed to be installed and operated in a CO2 incubator, so it has a compact and simple cantilever structure. In order to excite vibrations in each direction of orthogonal coordinate system, it has made from one stainless steel strip into a V-shaped vibrator, which is kinetically designed. Three piezoelectric ceramics were bonded on the vibrator, so that it is able to apply the three-dimensional vibration stimulation onto the cells on the culture dish. By using the developed stage, the effect of vibration stimulation was investigated morphologically. The experimental result shows that the vibration stimulation decreases the projected area and increases the slenderness ratio. In other words, the dynamic stimulation inhibits the progression of morphological change according to cell senescence. This might suggest that the stimulation affects gene expression pattern. Also, the most effective condition of vibration was horizontal direction of 10 Hz. It is found that the developed micro vibration stage is quite useful as a control device for cell culture.

Piezoelectric Micro Probe Device for Mechanical Stimulation and Its Detection for Living Cells

As one of the cellular responses to external mechanical stimulation, it is presumable that the cell adjusts the cytoskeletal mechanical strength globally as well as locally. However, methodologies to validate the hypothesis are extremely limited and expensive. In this study, a new micro probe device, utilizing dynamic response of a piezoelectric vibrator, is developed, which works not only as a sensor to evaluate local stiffness, but also as an actuator to enforce local mechanical stimulation onto cultured living adhesive cells. The second mode of vibration of a beam vibrator with clamped-free boundary condition is utilized because of its stability and large vibration displacement at the vibrating free end. In order to excite the second mode of vibration, a stainless-steel plate was used for the vibrator. Two pieces of piezoelectric ceramics, one for excitation and the other for detection, are bonded onto the vibrator. In order to have contact with a minute living cell and to enhance sensing capability, a newly designed microprobe having stepped cross section was set at the free end of the vibrator. The experimental setup consists the developed micro probe device, two FFT analyzers to drive the micro probe device sensing and actuation function, a three-dimensional micromanipulator to operate the micro probe device, an inverted phase contrast microscope and some assisting devices. Focusing on an osteoblastic feature, which is sensitive to external mechanical stimuli, normal human osteoblast (NHOst) was chosen as a specimen. As the result of the experiment, depending on the cytoskeletal condition, the cell showed different behaviors against vibration stimulations with same amplitude but different frequency. This suggests that the overall actin cytoskeleton has an importance to initiate the local cellular response.