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Image-based cell phenotyping is fundamental in both cell biology and medicine. As cells are dynamic systems, phenotyping based on static data is complemented by dynamic data extracted from time-dependent cell characteristics. We developed a label-free automatic tracking method for phase contrast images. We examined the possibility of using cell motility-based discrimination to identify different types of mesenchymal migration in invasive malignant cancer and non-cancer cells. These cells were cultured in plastic tissue culture vessels, using motility parameters from cell trajectories extracted with label-free tracking. Correlation analysis with these motility parameters identified characteristic parameters for cancer HT1080 fibrosarcoma and non-cancer 3T3-Swiss fibroblast cell lines. The parameter “sum of turn angles,” combined with the “frequency of turns” at shallow angles and “migration speed,” proved effective in highlighting the migration characteristics of these cells. It revealed differences in their mechanisms for generating effective propulsive forces. The requirements to characterize these differences included the spatiotemporal resolution of segmentation and tracking, capable of detecting polarity changes associated with cell morphological alterations and cell body displacement. With the segmentation and tracking method proposed here, a discrimination curve computed using quadratic discrimination analysis from the “sum of turn angles” and “frequency of turns below 30°” gave the best performance with a 94% sensitivity. Cell migration is a process related not only to cancer but also to tissue healing and growth. The proposed methodology is easy to use, enabling anyone without professional skills in image analysis, large training datasets, or special devices. It has the potential for application not only in cancer cell discrimination but also in a broad range of applications and basic research. Validating the expandability of this method to characterize cell migration, including the scheme of propulsive force generation, is an important consideration for future study.

The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo .

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction

Assessment of respiratory sounds by auscultation with a conventional stethoscope is subjective. We developed a continuous monitoring and visualization system that enables objectively and quantitatively visualizing respiratory sounds. We herein present two cases in which the system showed regional differences in the respiratory sounds. We applied our novel continuous monitoring and visualization system to evaluate respiratory abnormalities in patients with acute chest disorders. Respiratory sounds were continuously recorded to assess regional changes in respiratory sound volumes. Because we used this system as a pilot study, the results were not shown in real time and were retrospectively analyzed. Case 1 An 89-year-old woman was admitted to our hospital for sudden-onset respiratory distress and hypoxia. Chest X-rays revealed left pneumothorax; thus, we drained the thorax. After confirming that the pneumothorax had improved, we attached the continuous monitoring and visualization system. Chest X-rays taken the next day showed exacerbation of the pneumothorax. Visual and quantitative findings showed a decreased respiratory volume in the left lung after 3 h. Case 2 A 94-year-old woman was admitted to our hospital for dyspnea. Chest X-rays showed a large amount of pleural effusion on the right side. The continuous monitoring and visualization system visually and quantitatively revealed a decreased respiratory volume in the lower right lung field compared with that in the lower left lung field. Our newly developed continuous monitoring and visualization system enabled quantitatively and visually detecting regional differences in respiratory sounds in patients with pneumothorax and pleural effusion.

A Micro Electro-Mechanical System (MEMS) is a micro-sized mechatronic device based on semiconductor integrated-circuit manufacturing technology. MEMS technology is inherently a very powerful tool for researchers to manipulate and measure the microscopic biological components of biological tissues since their basic constituent units, cells and intracellular microstructures, are also micron or even submicron in size. MEMS technology applied to medicine and life sciences is called Bio-MEMS. Many Bio-MEMS devices and technologies have been developed in recent years, including microfluidic devices for cell culture, manipulation, and measurement, as well as lab-on-a-chip devices for chemical reactions and analyses on MEMS devices. This special issue consists of 13 papers: 1 review article, 1 letter, and 11 research papers. These papers include studies on mechanical stress on cells using MEMS devices, the control of cell functions through microfabrication techniques, cell measurement and in vivo microenvironment simulation using microfluidic devices, and basic research on wearable devices and self-assembling microstructures using MEMS technologies. The editorial committee members are confident that this special issue will make a significant contribution to the further development of Bio-MEMS. We sincerely appreciate the excellent contributions of the authors and the time and effort of the reviewers. We would also like to thank the editorial board of the Journal of Robotics and Mechatronics for their support of this special issue.

Migrating cells in vivo monitor the physiological state of an organism by integrating the physical as well as chemical cues in the extracellular microenvironment, and alter the migration mode, in order to achieve their unique function. The clarification of the mechanism focusing on the topographical cues is important for basic biological research, and for biomedical engineering specifically to establish the design concept of tissue engineering scaffolds. The aim of this study is to understand how cells sense and respond to the complex topographical cues in vivo by exploring in vitro analyses to complex in vivo situations in order to simplify the issue. Since the intracellular mechanical events at subcellular scales and the way of the coordination of these events are supposed to change in the migrating cells, a key to success of the analysis is a mechanical point of view with a particular focus of the subcellular mechanical events. We designed an experimental platform to explore the mechanical requirements in a migrating fibroma cell responding to micro-grooves. The micro-grooved structure is a model of gap structures, typically seen in the microenvironments in vivo. In our experiment, the contributions of actomyosin force generation can be spatially divided and analyzed in the cell center and peripheral regions. The analysis specified that rapid leading edge protrusion, and the cell body translocation coordinated with the leading edge protrusion are required for the turning response at a micro-groove.

To understand the mechanism regulating the spontaneous change in polarity that leads to cell turning, we quantitatively analyzed the dynamics of focal adhesions (FAs) coupling with the self-assembling actin cytoskeletal structure in Swiss 3T3 fibroblasts. Fluorescent images were acquired from cells expressing GFP-actin and RFP-zyxin by laser confocal microscopy. On the basis of the maximum area, duration, and relocation distance of FAs extracted from the RFP-zyxin images, the cells could be divided into 3 regions: the front region, intermediate lateral region, and rear region. In the intermediate lateral region, FAs appeared close to the leading edge and were stabilized gradually as its area increased. Simultaneously, bundled actin stress fibers (SFs) were observed vertically from the positions of these FAs, and they connected to the other SFs parallel to the leading edge. Finally, these connecting SFs fused to form a single SF with matured FAs at both ends. This change in SF organization with cell retraction in the first cycle of migration followed by a newly formed protrusion in the next cycle is assumed to lead to cell turning in migrating Swiss 3T3 fibroblasts.

Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.

Electrospun gelatin nanofibers are effective tissue engineering scaffolds with high biocompatibility and cell adhesion activity. In gelatin electrospinning, fluorinated alcohols, which are irritants, and acidic organic solvents are used as solvents to prevent gelation. This study established a technique to embed protein reagents into the nanofibers using mild solvents. From 22 mixtures of 50% organic solvent–50% H 2 O, less denaturing neutral dipolar aprotic solvents (specifically N , N -dimethylacetamide, N , N -dimethylformamide and N -methyl-2-pyrrolidone), were screened to assess their suitability for use in electrospinning of gelatin nanofiber scaffolds by their ability to maintain gelatin in a sol state at room temperature. By selecting the solvents and their concentrations, gelatin nanofibers were electrospun with different structures from a thick, wide, porous nanofibrous structure to a thin, fine, nanofibrous mesh structure. Swiss 3T3 fibroblasts grew well on the gelatin nanofibers. In particular, some cells showed in vivo -like spindle morphologies on the thick porous nanofibers using N , N -dimethylacetamide. Additionally, as a model protein reagent, alkaline phosphatase was embedded in the gelatin nanofibers while maintaining high activity. Considering these results, the gelatin nanofibers in this study are expected to provide effective structural and chemical cues and will be useful for tissue engineering and regenerative medicine. Gelatin nanofibers were electrospun using mild N,N -dimethylacetamide, N,N -dimethylformamide and N -methyl-2-pyrrolidone. Various gelatin nanofibers were fabricated, ranging from a thick, wide, porous nanofibrous structure to a thin, fine, nanofibrous mesh structure, depending on the solvents and their concentrations. In particular, Swiss 3T3 fibroblasts exhibited in vivo -like spindle morphologies on the thick nanofibers. A model protein, alkaline phosphatase, embedded in the gelatin nanofibers maintained high activity.