Yuta Kurashina

Effective cell collection method using collagenase and ultrasonic vibration

This study proposes a novel cell collection method based on collagenase treatment and ultrasonic vibration. The method collects calf chondrocytes from a reusable metal cell culture substrate. To develop our concept, we calculated the natural vibration modes of the cell culture substrate by a finite element method, and conducted eigenvalue and piezoelectric-structural analyses. Selecting the first out-of-plane vibration mode of the substrate, which has a single nodal circle, we designed and fabricated the cell collection device. The excited vibration mode properly realized our intentions. We then evaluated the cell collection ratio and the growth response, and observed the morphology of the collected cells. The collagenase and ultrasonic vibration treatment collected comparable numbers of cells to conventional trypsin and pipetting treatment, but improved the proliferating cell statistics. Morphological observations revealed that the membranes of cells collected by the proposed method remain intact; consequently, the cells are larger and rougher than cells collected by the conventional method. Therefore, we present a promising cell collection method for adhesive cell culturing process.

Efficient Subculture Process for Adherent Cells by Selective Collection Using Cultivation Substrate Vibration

Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture—without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 µm, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72-h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.

Cell manipulation by nodal circle resonance vibration of a cell cultivation substrate

In this paper, we propose a novel cell culture method to generate an organ without scaffold. The concept of our study is to apply the principle of Chladnis figures in cell manipulation. To confirm this concept, we developed cell cultivation device that can excite resonance vibration of the cell cultivation substrate. After the fabrication of the device, we estimated the resonance frequency and vibration amplitude distribution of our device. Since the fabricated device successfully produced the designed vibration mode, we conducted cell manipulation experiment to confirm our concept. In our experiment, we varied the initial number of cells that were seeded into our device. Cells were manipulated by resonance vibration for 120 min. After the manipulation, we checked cell distribution on the substrate. As a result, cells were successfully manipulated by the resonance vibration when the initial number of cells was appropriate.

Growth control of leaf lettuce with exposure to underwater ultrasound and dissolved oxygen supersaturation

The growth rate of vegetables in plant factories can be regulated by environmental factors including light, temperature, and chemicals, which might give rise to mutation in leaf health. Here, we aim to devise a new way that allows for controlling the growth rate of plants in hydroponics as well as maintaining the product quality; we apply underwater ultrasound and dissolved oxygen supersaturation as external stimuli to plants. As an example, we examine the growth of leaf lettuce in hydroponics with exposure to 28-kHz ultrasound and dissolved oxygen supersaturation up to 36 mg/L at 20 °C. Our results show that exposure to the ultrasound of peak-to-peak pressure at 20 kPa or larger works as the growth inhibitor of the leaves and the roots, while the oxygen supersaturation as the growth promoter, without any degradation of chlorophyll in the leaves. This suggests that these external stimuli can be used in the growth control system of plant factories.

Tactile estimation of molded plastic plates based on the estimated impulse responses of mechanoreceptive units

This study proposes a tactile estimation method of molded plastic plates based on human tactile perception characteristics. Plastic plates are often used in consumer products. The tactile evaluation plays an important role in product development. However, physical quantities not taking into account human tactile perception have been employed in previous tactile estimation procedures. Hence, in this study, we adopted the vibrational thresholds of the mechanoreceptive units—FA I, FA II, SA I and SA II—for stimuli detection and developed a tactile estimation method for plastic plates that clarified the mechanoreceptive units related to tactile sensation. The developed tactile sensor consists of a base and a silicone rubber pad that contains strain gauges in it. We detected vibration during touch by the sensor and calculated the estimation of the firing values of the cutaneous mechanoreceptors, which are the essential data obtained by humans during tactile perception, in comparison to the amplitude spectrum of the vibration with the threshold amplitude of each mechanoreceptive unit. Simultaneously, we calculated the relationship between the normal and tangential forces recorded while the sensor ran over the samples. As a result of stepwise linear regression analysis using these values as explanatory variables, the evaluation scores for Soft were successfully estimated using the firing value of FA II and the relationship between normal/tangential forces, and the evaluation scores for Rough were estimated using the SA I firing value.

A Method for Collecting Single Cell Suspensions Using an Ultrasonic Pump

The presence of cell aggregates in cell suspensions may reduce cell culture efficiency because they can induce apoptosis and inhibit proliferation. To avoid this problem, this study proposes a novel method for collecting single cell suspensions from culture chambers for subculture using an ultrasonic pump driven by the squeeze film effect. First, we developed a cell culture device consisting of a cell culture substrate with a piezoelectric ceramic disk glued to the back, so that we can elicit resonance vibration of the substrate. A glass pipe is then placed vertically against the cell culture substrate with a slight gap (corresponding to cell diameter) between the pipe and the substrate. By exciting an out-of-plane resonance vibration of the cell culture substrate, we can collect a cell suspension from the cell culture chamber. Since the gap distance between the glass pipe and the cell culture substrate corresponds to cell diameter, the collected cell suspension only contains single cells. We evaluated the capability of the developed cell suspension pumping system and the proliferation of the collected cells with C2C12 myoblast cells. The ratio of single cells in the cell suspension was improved by up to 9.6% compared with that of suspensions collected by the control method (traditional pipetting). Moreover, after cultivating the collected cells for 72 hr, the cells collected by our method proliferated 13.6% more than those collected by the control method. These results suggest that the proposed method has great potential for improving the cultivation efficiency of adhesive cell culture.

Cell patterning method on a clinically ubiquitous culture dish using acoustic pressure generated from resonance vibration of a disk-shaped ultrasonic transducer

Cell patterning methods have been previously reported for cell culture. However, these methods use inclusions or devices that are not used in general cell culture and that might affect cell functionality. Here, we report a cell patterning method that can be conducted on a general cell culture dish without any inclusions by employing a resonance vibration of a disk-shaped ultrasonic transducer located under the dish. A resonance vibration with a single nodal circle patterned C2C12 myoblasts into a circular shape on the dish with 10-min exposure of the vibration with maximum peak–peak amplitude of 10 μm

Effect of underwater ultrasound exposure on growth of plant roots and leaves

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Enzyme‐free cell detachment mediated by resonance vibration with temperature modulation

. Furthermore, the relationship between the amplitude distribution of the transducer and the cell density in the patterned sample could be expressed as a linear function, and there was a clear threshold of amplitude for cell adhesion. To evaluate the cell function of the patterned cells, we conducted proliferation and protein assays at 120-h culture after patterning. Our results showed that the cell proliferation rate did not decrease and the expression of cellular proteins was unchanged. Thus, we conclude, this method can successfully pattern cells in the clinically ubiquitous culture dish, while maintaining cell functionality.

Proliferation of calf chondrocyte on stainless-steel surfaces with different microtopography

Growth control is an important technique in plant markets, for appropriate shipping time of plants (e.g., flowers and fruits) allows for reducing production costs for post harvesting storage and pesticide. It is favorable to control plant growth without any chemicals; physical stresses are attractive candidates for this purpose. Here, we propose an ultrasound-based technique to control plant growth.