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.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

The degeneration of adipocyte has been reported to cause obesity, metabolic syndrome, and other diseases. To treat these diseases, an effective in vitro evaluation and drug-screening system for adipocyte culture is required. The objective of this study is to establish an in vitro three-dimensional cell culture system to enable the monitoring of lipid accumulation by measuring electrical impedance, and to determine the relationship between the impedance and lipid accumulation of adipocytes cultured three dimensionally. Consequently, pre-adipocytes, 3T3-L1 cells, were cultured and differentiated to the adipocytes in our culture system, and the electrical impedance of the three-dimensional adipocyte culture at a high frequency was related to the lipid accumulation of the adipocytes. In conclusion, the lipid accumulation of adipocytes could be evaluated in real time by monitoring the electrical impedance during in vitro culture.

Fabrication of three-dimensional tissues using living cells is a promised approach for drug screening experiment and in vitro disease modeling. To study a physiological neuronal function, three-dimensional cell patterning and construction of neuronal cell network were required. In this study, we proposed a three-dimensional cell drawing methodology in hydrogel to construct the three-dimensional neuronal cell network. PC-12 cells, which were used as neuronal cell differentiation model, were dispensed into a collagen hydrogel using a micro injector with a three-dimensional position control. To maintain the three-dimensional position of cells, atelocollagen was kept at sol-gel transition state during cell dispensing. As the results, PC-12 cells were patterned in the atelocollagen gel to form square pattern with different depth. In the patterned cellular lines, PC-12 cells elongated neurites and form a continuous cellular network in the atelocollagen gel. It was suggested that our three-dimensional cell drawing technology has potentials to reconstruct three-dimensional neuronal networks for an investigation of physiological neuronal functions.

Engineering of the skeletal muscles has attracted attention for the restoration of damaged muscles from myopathy, injury, and extraction of malignant tumors. Reconstructing a three-dimensional muscle using living cells could be a promising approach. However, the regenerated tissue exhibits a weak construction force due to the insufficient tissue maturation. The purpose of this study is to establish the reconstruction system for the skeletal muscle. We used a cell-laden core-shell hydrogel microfiber as a three-dimensional culture to control the cellular orientation. Moreover, to mature the muscle tissue in the microfiber, we also developed a custom-made culture device for imposing cyclic stretch stimulation using a motorized stage and the fiber-grab system. As a result, the directions of the myotubes were oriented and the mature myotubes could be formed by cyclic stretch stimulation.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Malignant melanoma in the plantar surface of the foot is subjected to various mechanical stimuli generated by daily human activity such as walking. Some studies have reported that mechanical compression affects the development and progression of melanoma. However, little is known about how mechanical compression affects the behavior of malignant melanoma cells in a physiological condition due to the complexity of the invasion mechanisms. In this study, we developed an in vitro three-dimensional cell culture device using microporous membrane in order to evaluate the effects of mechanical compression on the invasion process of malignant melanoma. Our results suggest that the invasion of melanoma cells under the compressive stress for 8 h of culture was promoted with the elongation of F-actin filaments compared to control groups, whereas there was no significant difference between both groups at 32 h of culture, with increasing cell death associated with promoting melanin synthesis. The results of this study contribute to the elucidation of the invasion mechanisms of malignant melanoma caused by mechanical stimulation.

To generate three-dimensional tissue in vitro, promoting vasculogenesis in cell aggregates is an important factor. Here, we found that ultrasound promoted vasculogenesis of human umbilical vein endothelial cells (HUVECs). Promotion of HUVEC network formation and lumen formation were observed using our method. In addition to morphological evaluations, protein expression was quantified by western blot assays. As a result, expression of proteins related to vasculogenesis and the response to mechanical stress on cells was enhanced by exposure to ultrasound. Although several previous studies have shown that ultrasound may promote vasculogenesis, the effect of ultrasound was unclear because of unregulated ultrasound, the complex culture environment, or two-dimensional-cultured HUVECs that cannot form a lumen structure. In this study, regulated ultrasound was propagated on three-dimensional-monocultured HUVECs, which clarified the effect of ultrasound on vasculogenesis. We believe this finding may be an innovation in the tissue engineering field.

Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Decellularized tissues are promising materials that mainly consist of extracellular matrices (ECMs) obtained by removing all cells from organs and tissues. High hydrostatic pressure (HHP) has been used for decellularization to remove cells physically from organs or tissues rather than by chemical methods. However, ultrahigh pressure induces denaturation of the ECM structure. In this study, we examined the effects of cyclic HHP at low and high pressures on the cell membrane structure to establish a novel decellularization method that enables decellularization without the denaturation of the ECM. A decellularization device using cyclic HHP (maximum pressure: 250 MPa, cycle number: 5) was developed. NB1RGB cell suspension was injected into a plastic bag to be subjected to cyclic HHP. After applying cyclic HHP, the amount of DNA inside the cells and the morphological changes of the cells were evaluated. As a result, the amount of DNA inside the cells decreased after the cyclic HHP compared to the static HHP. In addition, cyclic HHP was suggested to promote the destruction of the cell and nuclear membrane. In conclusion, it was revealed that the cell structure could be denatured and destroyed by cyclic HHP at a lower level than that of previous approaches.

Recently, automated cell culture devices have become necessary for cell therapy applications. The maintenance of cell functions is critical for cell expansion. However, there are risks of losing these functions, owing to disturbances in the surrounding environment and culturing procedures. Therefore, there is a need for a non-invasive and highly accurate evaluation method for cell phenotypes. In this study, we focused on an automated discrimination technique using image processing with a deep learning algorithm. This study aimed to clarify the effects of the optical magnification of the microscope and cell size in each image on the discrimination accuracy for cell phenotypes and morphologies. Myoblast cells (C2C12 cell line) were cultured and differentiated into myotubes. Microscopic images of the cultured cells were acquired at magnifications of 40× and 100×. A deep learning architecture was constructed to discriminate between undifferentiated and differentiated cells. The discrimination accuracy exceeded 90% even at a magnification of 40× for well-developed myogenic differentiation. For the cells under immature myogenic differentiation, a high optical magnification of 100× was required to maintain a discrimination accuracy over 90%. The microscopic optical magnification should be adjusted according to the cell differentiation to improve the efficiency of image-based cell discrimination.

Recently, many studies have focused on the repair and regeneration of damaged articular cartilage using tissue engineering. In tissue engineering therapy, cells are cultured in vitro to create a three-dimensional (3-D) tissue designed to replace the damaged cartilage. Although tissue engineering is a useful approach to regenerating cartilage, mechanical anisotropy has not been reconstructed from a cellular organization level. This study aims to create mechanically anisotropic cartilaginous tissue using dielectrophoretic cell patterning and gel-sheet lamination. Bovine chondrocytes were patterned in a hydrogel to form line-array cell clusters via negative dielectrophoresis (DEP). The results indicate that the embedded chondrocytes remained viable and reconstructed cartilaginous tissue along the patterned cell array. Moreover, the agarose gel, in which chondrocytes were patterned, demonstrated mechanical anisotropy. In summary, our DEP cell patterning and gel-sheet lamination techniques would be useful for reconstructing mechanically anisotropic cartilage tissues.

Static mechanical compression is a biomechanical factor that affects the progression of melanoma cells. However, little is known about how dynamic mechanical compression affects the progression of melanoma cells. In the present study, we show that mechanical intermittent compression affects the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results suggest that intermittent compression with a cycle of 2 h on/2 h off could suppress the progression rate of melanoma cells by suppressing the elongation of F-actin filaments and mRNA expression levels related to collagen degradation. In contrast, intermittent compression with a cycle of 4 h on/4 h off could promote the progression rate of melanoma cells by promoting cell proliferation and mRNA expression levels related to collagen degradation. Mechanical intermittent compression could therefore affect the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results contribute to a deeper understanding of the physiological responses of melanoma cells to dynamic mechanical compression.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Cancer research is increasingly focused on discovering strategies to induce cancer cell apoptosis without affecting surrounding normal cells. One potential biocompatible method is mechanical vibration, which has been developed as part of the emerging field of mechanomedicine. Previous studies of mechanical vibration have employed high-frequency vibration, which damages healthy cells. In this study, we examined the effects of brief (1 h) low-frequency (20 Hz) mechanical vibration on glucose consumption and survival (apoptosis, necrosis, HMGB1 release) of the human epidermoid carcinoma cell line A431. We found that apoptosis, but not necrosis, was significantly increased at 48 h after mechanical vibration compared with cells maintained in static culture. In keeping with this, extracellular release of HMGB1, a necrosis marker, was lower in cultures of A431 cells subjected to mechanical vibration compared with control cells. Glucose consumption was increased in the first 24 h after mechanical vibration but returned to control levels before the onset of apoptosis. Although the precise intracellular mechanisms by which low-frequency mechanical vibration triggers apoptosis of A431 cells is unknown, these results suggest a possible role for metabolic pathways. Mechanical vibration may thus represent a novel application of mechanomedicine to cancer therapy.

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.

Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to "native" hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages.

Hydrogen production techniques based on solar-water splitting have emerged as carbon-free energy systems. Many researchers have developed highly efficient thin-film photoelectrochemical (PEC) devices made of low-cost and earth-abundant materials. However, solar water splitting systems suffer from short lifetimes due to catalyst instability that is attributed to both chemical dissolution and mechanical stress produced by hydrogen bubbles. A recent study found that the nanoporous hydrogel could prevent the structural degradation of the PEC devices. In this study, we investigate the protection mechanism of the hydrogel-based overlayer by engineering its porous structure using the cryogelation technique. Tests for cryogel overlayers with varied pore structures, such as disconnected micropores, interconnected micropores, and surface macropores, reveal that the hydrogen gas trapped in the cryogel protector reduce shear stress at the catalyst surface by providing bubble nucleation sites. The cryogelated overlayer effectively preserves the uniformly distributed platinum catalyst particles on the device surface for over 200 h. Our finding can help establish semi-permanent photoelectrochemical devices to realize a carbon-free society.

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.