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.

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.

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.

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.

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, 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.

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.

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.

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.

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.

In this study, we proposed an in vitro tumor model to simulate the mechanical microenvironment and investigate the effect of compressive stress on the invasion process of malignant tumors. It has been pointed out that the biomechanical environment, as well as the biochemical environment, could affect the transformation of cancer cell migration, invasion, and metastasis. We hypothesized that the solid stress caused by the exclusion of surrounding tissue could transform tumor cells from noninvasive to invasive phenotypes. Colorectal cell spheroids were embedded and cultured in agarose gels of varying concentrations to simulate the earliest stages of tumor formation and invasion. The spheroids embedded in gels at higher concentrations showed peculiar growth after 72 h of culture, and the external compressive loading imposed on them caused peculiar growth even in the gels at lower concentrations. In conclusion, the mechanical microenvironment caused the transformation of tumor cell phenotypes, promoting the growth and invasion of tumor cell spheroids.

Pluripotent stem cells (PSCs) such as embryonic stem cells and induced PSCs can differentiate into all somatic cell types such as cardiomyocytes, nerve cells, and chondrocytes. However, PSCs can easily lose their pluripotency if the culture process is disturbed. Therefore, cell sorting methods for purifying PSCs with pluripotency are important for the establishment and expansion of PSCs. In this study, we focused on dielectrophoresis (DEP) to separate cells without fluorescent dyes or magnetic antibodies. The goal of this study was to establish a cell sorting method for the purification of PSCs based on their pluripotency using DEP and a flow control system. The dielectrophoretic properties of mouse embryonic stem cells (mESCs) with and without pluripotency were evaluated in detail, and mESCs exhibited varying frequency dependencies in the DEP response. Based on the variance in DEP properties, mixed cell suspensions of mESCs can be separated according to their pluripotency with an efficacy of approximately 90%.