Spatiotemporal single-cell tracking analysis in 3D tissues to reveal heterogeneous cellular response to mechanical stimuli.

Mechanical stimuli have been recognized as important for tissue maturation, homeostasis and constructing engineered three-dimensional (3D) tissues. However, we know little about the cellular mechanical response in tissues that could be considerably heterogeneous and spatiotemporally dynamic due to the complex structure of tissues. Here, we report a spatiotemporal single-cell tracking analysis of in vitro 3D tissues under mechanical stretch, to reveal the heterogeneous cellular behavior by using a developed stretch and optical live imaging system.

Multilayered Gel-Spotting Device for Reconstruction of Hair Follicle-like Microstructure.

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 . 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.

Three-Dimensional Cell Drawing Technique in Hydrogel Using Micro Injection System.

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.

Effect of Compressive Stress in Tumor Microenvironment on Malignant Tumor Spheroid Invasion Process.

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.

Efficient Cell Impedance Measurement by Dielectrophoretic Cell Accumulation and Evaluation of Chondrogenic Phenotypes.

The quantitative and functional analyses of cells are important for cell-based therapies. In this study, to establish the quantitative cell analysis method, we propose an impedance measurement method supported by dielectrophoretic cell accumulation. An impedance measurement and dielectrophoresis device was constructed using opposing comb-shaped electrodes.

Influences of Microscopic Imaging Conditions on Accuracy of Cell Morphology Discrimination Using Convolutional Neural Network of Deep Learning.

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.

Purification of pluripotent embryonic stem cells using dielectrophoresis and a flow control system.

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.

Efficient Decellularization by Application of Moderate High Hydrostatic Pressure with Supercooling Pretreatment.

Decellularized tissues are considered superior scaffolds for cell cultures, preserving the microstructure of native tissues and delivering many kinds of cytokines. High hydrostatic pressure (HHP) treatment could remove cells physically from biological tissues rather than chemical methods. However, there are some risks of inducing destruction or denaturation of extracellular matrices (ECMs) at an ultrahigh level of HHP.

Development of accurate temperature regulation culture system with metallic culture vessel demonstrates different thermal cytotoxicity in cancer and normal cells.

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.

Dielectrophoretic Micro-Organization of Chondrocytes to Regenerate Mechanically Anisotropic Cartilaginous Tissue.

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.

Mechanical Intermittent Compression Affects the Progression Rate of Malignant Melanoma Cells in a Cycle Period-Dependent Manner.

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.

UV/ozone surface modification combined with atmospheric pressure plasma irradiation for cell culture plastics to improve pluripotent stem cell culture.

Culturing pluripotent stem cells effectively requires substrates coated with feeder cell layers or cell-adhesive matrices. It is difficult to employ pluripotent stem cells as resources for regenerative medicine due to risks of culture system contamination by animal-derived factors, or the large costs associated with the use of adhesive matrices. To enable a coating-free culture system, we focused on UV/ozone surface modification and atmospheric pressure plasma treatment for polystyrene substrates, to improve adhesion and proliferation of pluripotent stem cells.

Fundamental Study of Decellularization Method Using Cyclic Application of High Hydrostatic Pressure.

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.

Continuous ES/Feeder Cell-Sorting Device Using Dielectrophoresis and Controlled Fluid Flow.

Pluripotent stem cells (PSCs) are considered as being an important cell source for regenerative medicine. The culture of PSCs usually requires a feeder cell layer or cell adhesive matrix coating such as Matrigel, laminin, and gelatin. Although a feeder-free culture using a matrix coating has been popular, the on-feeder culture is still an effective method for the fundamental study of regenerative medicine and stem cell biology.

Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration.

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.

Cost-effective culture of human induced pluripotent stem cells using UV/ozone-modified culture plastics with reduction of cell-adhesive matrix coating.

Human induced pluripotent stem cells (hiPSCs) are considered to be one of the most promising cell resources for regenerative medicine. HiPSCs usually maintain their pluripotency when they are cultured on feeder cell layers or are attached to a cell-adhesive extracellular matrix. In this study, we developed a culture system based on UV/ozone modification for conventional cell culture plastics to generate a suitable surface condition for hiPSCs.

Effect of Mechanical Compression on Invasion Process of Malignant Melanoma Using In Vitro Three-Dimensional Cell Culture Device.

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.

Evaluation of Lipid Accumulation Using Electrical Impedance Measurement under Three-Dimensional Culture Condition.

The degeneration of adipocyte has been reported to cause obesity, metabolic syndrome, and other diseases. To treat these diseases, an effective evaluation and drug-screening system for adipocyte culture is required. The objective of this study is to establish an 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.

Effect of Cyclic Stretch on Tissue Maturation in Myoblast-Laden Hydrogel Fibers.

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.

Feeder-free culture for mouse induced pluripotent stem cells by using UV/ozone surface-modified substrates.

Pluripotent stem cells (PSCs), especially induced PSCs (iPSCs), have great potential for regenerative medicine. Conventionally, PSCs are cultured and expanded efficiently on feeder cell layers or on cell-adhesive matrices. Large-scale iPSC expansion in an undifferentiated state without laborious culturing procedures and high manufacturing costs for the adhesive matrix is urgently required to integrate iPSCs into therapeutic applications.

Improvement of adhesion and proliferation of mouse embryonic stem cells cultured on ozone/UV surface-modified substrates.

Culturing pluripotent stem cells effectively requires feeder cell layers or cell adhesion matrix coating. However, the feeder cell layers or animal-derived factors have to be removed to apply the pluripotent stem cells as resources for regenerative medicine. To enable xeno-free culture conditions, we focused on the UV/ozone surface treatment technique for polystyrene cell culture substrates to improve the adhesion and proliferation of pluripotent stem cells.

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.

Evaluation of negative fixed-charge density in tissue-engineered cartilage by quantitative MRI and relationship with biomechanical properties.

Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties.

Influence of cartilaginous matrix accumulation on viscoelastic response of chondrocyte/agarose constructs under dynamic compressive and shear loading.

A method has been developed to restore cartilage defects by culturing autologous chondrocytes to create a three dimensional tissue and then implanting the cultured tissue. In this kind of approach, it is important to characterize the dynamic mechanical behavior of the regenerated cartilaginous tissue, because these tissues need to bear various dynamic loadings in daily life. The objectives of this study were to evaluate in detail the dynamic viscoelastic responses of chondrocyte-seeded agarose gel cultures in compression and torsion (shear) and to determine the relationships between these mechanical responses and biochemical composition.

Feasibility of noninvasive evaluation of biophysical properties of tissue-engineered cartilage by using quantitative MRI.

The application of tissue-engineered cartilage in a clinical setting requires a noninvasive method to assess the biophysical and biochemical properties of the engineered cartilage. Since articular cartilage is composed of 70-80% water and has dense extracellular matrixes (ECM), it is considered that the condition of the water molecules in the tissue is correlated with its biomechanical property. Therefore, magnetic resonance imaging (MRI) represents a potential approach to assess the biophysical property of the engineered cartilage.