A reel mechanism-based robotic colonoscope with high safety and maneuverability.

BACKGROUND: At present, the colonoscopy is the most common method of screening for colorectal cancer. However, endoscopists still encounter difficulties with intubation, primarily due to the structural diversity (e.g., path, shape, and size) and viscoelasticity of the colon. Therefore, well-trained, skillful operators are required to overcome these factors and operate colonoscopes without harming patients. OBJECTIVES: In our previous work, we presented a reel mechanism-based robotic colonoscope designed to mitigate the difficulties of conventional colonoscopies. Although we reported excellent mobile performance with respect to the robot, we did not provide an in-depth discussion concerning patient safety. Therefore, in this article, we propose a method of improving robot safety, and this is verified by investigating the static and dynamic forces acting on the colon. In addition, the maneuverability and safety of the robot in the in vitro condition are evaluated. METHODS: The safety solution is provided by covering the robot's legs with silicone. To evaluate the results, the reaction force according to leg deformation is measured. Then, the force transmitted to the colon is also measured when the robot moves through various environments. Finally, a mobility test on an excised porcine colon is performed to simultaneously verify the robot's maneuverability and safety. RESULTS: We verify that the static and dynamic force acting on the colon is less than the burst force of a human colon. In addition, the maneuverability of the robotic colonoscope shows reliable locomotion performance even with the soft material covering the legs; it has forward velocities of 9.552 ± 1.940 mm/s on a flat path. CONCLUSION: Owing to the reliable locomotion mechanism with the safety-securing silicone, the robot achieves high and reliable maneuverability without any scratches or perforations to the porcine colon.

Molecular analysis of the inhibitory effect of N-acetyl-L-cysteine on the proliferation and invasiveness of pancreatic cancer cells.

Preliminary studies have suggested that the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) may be effective in inhibiting the growth of pancreatic cancer cells. In-depth cellular and molecular analyses were carried out to determine NAC's mode of action in inhibiting the growth of a well-characterized pancreatic cancer cell line (AsPC-1). Standardized assays were used to monitor cellular growth, apoptosis, levels of ROS, cellular senescence, migration, and invasiveness. Cell stiffness was measured using atomic force microscopy. Gene expression was monitored by quantitative PCR. NAC significantly inhibits the growth and metastatic potential of AsPC-1 cells by inducing cell-cycle arrest in G1 and subsequent cellular senescence and decreased invasiveness. These anticancer properties are associated with an unexpected increase in the intracellular concentrations of ROS. NAC does not decrease the susceptibility of AsPC-1 cells to the anticancer drugs gemcitabine, mitomycin C, and doxorubicin. NAC-induced changes in gene expression are consistent with the onset of mesenchymal-to-epithelial transition. In conclusion, our findings indicate that NAC induces an integrated series of responses in AsPC-1 cells that make it a highly promising candidate for development as a pancreatic cancer therapeutic.

A Systematic Review and Meta-analysis of the Impact of Radiation-Related Lymphopenia on Outcomes in High-Grade Gliomas.

Supriya MallickIntroduction Malignant gliomas are the most common primary malignant brain tumors and are typically treated with maximal safe surgical resection followed by chemoradiation. One of the unintended effects of radiation is depletion of circulating lymphocyte pool, which has been correlated with inferior overall survival outcomes. Methods A comprehensive and systematic searches of the PubMed, Cochrane Central, and Embase databases were done to assess the studies that have reported radiation-related lymphopenia in high-grade gliomas. Hazard ratios (HRs), odds ratios (OR), and mean differences were represented with Forest plots comparing patients with severe lymphopenia and no severe lymphopenia. Review Manager Version 5.3 (The Nordic Cochrane Centre, Copenhagen, Denmark) was used for the analysis. Results Nineteen studies were included in the final systematic review and 12 studies were included in the meta-analysis. The odds of developing severe lymphopenia were 0.39 (95% CI:0.19, 0.81, I 2 = 94%, p = 0.01). Patients with severe lymphopenia were at increased risk of death with a pooled HR = 2.19 (95% CI: 1.70, 2.83, I 2 = 0%, p <0.00001) compared to patients with no severe lymphopenia. The mean difference in survival between patients with severe lymphopenia and no severe lymphopenia was -6.72 months (95% CI: -8.95, -4.49, I 2 = 99%, p <0.00001), with a better mean survival in the no severe lymphopenia group. Conclusion Radiation-induced severe lymphopenia was associated with poor overall survival and increased risk of death. Photon therapy, larger planning target volume, higher brain dose, higher hypothalamus dose, and female gender were associated with increased risk of severe lymphopenia.

Adoptive therapy with amyloid-ß specific regulatory T cells alleviates Alzheimer's disease.

Rationale: Neuroinflammation is a primary feature of Alzheimer's disease (AD), for which an increasing number of drugs have been specifically developed. The present study aimed to define the therapeutic impact of a specific subpopulation of T cells that can suppress excessive inflammation in various immune and inflammatory disorders, namely, CD4+CD25+Foxp3+ regulatory T cells (Tregs). Methods: To generate Aß antigen-specific Tregs (Aß+ Tregs), Aß 1-42 peptide was applied in vivo and subsequent in vitro splenocyte culture. After isolating Tregs by magnetic bead based purification method, Aß+ Tregs were adoptively transferred into 3xTg-AD mice via tail vein injection. Therapeutic efficacy was confirmed with behavior test, Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry staining (IHC). In vitro suppression assay was performed to evaluate the suppressive activity of Aß+ Tregs using flow cytometry. Thy1.1+ Treg trafficking and distribution was analyzed to explore the infused Tregs migration into specific organs in an antigen-driven manner in AD mice. We further assessed cerebral glucose metabolism using 18F-FDG-PET, an imaging approach for AD biological definition. Subsequently, we evaluated the migration of Aß+ Tregs toward Aß activated microglia using live cell imaging, chemotaxis, antibody blocking and migration assay. Results: We showed that Aß-stimulated Tregs inhibited microglial proinflammatory activity and modulated the microglial phenotype via bystander suppression. Single adoptive transfer of Aß+ Tregs was enough to induce amelioration of cognitive impairments, Aß accumulation, hyper-phosphorylation of tau, and neuroinflammation during AD pathology. Moreover, Aß-specific Tregs effectively inhibited inflammation in primary microglia induced by Aß exposure. It may indicate bystander suppression in which Aß-specific Tregs promote immune tolerance by secreting cytokines to modulate immune responses during neurodegeneration. Conclusions: The administration of Aß antigen-specific regulatory T cells may represent a new cellular therapeutic strategy for AD that acts by modulating the inflammatory status in AD.

Droplet-based magnetically activated cell separation: analysis of separation efficiency based on the variation of flow-induced circulation in a pendent drop.

Under the assumption that separation efficiencies are mainly affected by the velocity of flow-induced circulation due to buffer injection in a pendent drop, this paper describes an analysis of the separation efficiency of a droplet-based magnetically activated cell separation (DMACS) system. To investigate the velocity of the flow-induced circulation, we supposed that numerous flows in a pendent drop could be considered as a "theoretically normalized" flow (or conceptually normalized flow, CNF) based on the Cauchy-Goursat theorem. With the morphological characteristics (length and duration time) of a pendent drop depending on the initial volume, we obtained the velocities of the CNF. By measuring the separation efficiencies for different initial volumes and by analyzing the separation efficiency in terms of the velocity of the CNF, we found that the separation efficiencies (in the case of a low rate of buffer injection; 5 and 15 microl x min(-1)) are mainly affected by the velocity of the CNF. Moreover, we confirmed that the phenomenological features of a pendent drop cause a fluctuation of its separation efficiencies over a range of specific volumes (initial volumes ranging from 40 to 80 microl), because of the "sweeping-off" phenomenon, that is, positive cells gathered into the positive fraction are forced to move away from the magnetic side by flow-induced circulation due to buffer injection. In addition, from the variation of the duration time, that is, the interval between the beginning of injection of the buffer solution and the time at which a pendent drop detaches, it could also be confirmed that a shorter duration time leads to decrease of the number of positive cells in negative fraction regardless of the rate of buffer injection (5, 15, and 50 microl x min(-1)). Therefore, if a DMACS system is operated with a 15 microl x min(-1) buffer injection flow rate and an initial volume of 80 microl or more, we would have the best efficiency of separation in the negative fraction.

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS).

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V(pp). Finally, under the applied voltage of 48 MHz-8 V(pp) and a flow rate of 290 microm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.

Gravity and rheotaxis based sperm sorting device employing a cam-actuated pipette mechanism.

Until now, a swim-up or microchip-based method has been mainly utilized for separating normal sperm for use in assisted reproductive technology. However, it requires excessive sorting time due to preprocessing and collects a limited number of motile sperms. To improve this process, we propose a gravity-fed high motility sperm sorting device that utilizes the rheotaxis of sperm, which minimizes separation time and improves throughput. The device features a mesoscale microfluidic channel to maximize the throughput, and an outlet at the bottom is configured to control the fluid velocity in the channel by using gravity. To control and automate semen injection and suction of the sorted sperm, a pipette controller using a cam was fabricated. After constructing the system, a sorting experiment was performed using canine semen to confirm the separation efficiency. After injecting the semen in the channel, the delay time between injection and suction was measured and the relative improvement of the index of motility was investigated according to measured delay time. As a result of repeated experiments, it was confirmed that the highest improvement was obtained at a delay time of 80 s, and the mean velocity, %motility, MI, and motile sperm rates were improved by 8.94%, 32.58%, 35.48%, and 21.99%, respectively.

Micro pumping with cardiomyocyte-polymer hybrid.

This paper presents a hybrid micropump actuated by the up-down motion of a dome shaped cell-polymer membrane composite. The contractile force induced from self-beating cardiomyocytes cultured on the membrane causes shrinkage and relaxation of a microchamber, leading to a flow in a microchannel. Flow direction is controlled by the geometry of diffuser/nozzle in the microchannel. The fabrication process is noninvasive to cells, thus, cardiomyocytes can robustly maintain their activity for a long time. The fluid motion in the microchannel was monitored by tracking 2 microm polystyrene beads. A net flow rate of 0.226 nl min(-1) was obtained in our microscale device. Our device demonstrates a unique performance of a cell-microdevice hybrid lab-on-a-chip that does not require any external power source, preventing electrical or heat shock to analytes.

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference.

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.

Establishment of a fabrication method for a long-term actuated hybrid cell robot.

We developed a novel method to fabricate a crab-like microrobot that can actuate for a long period in a physiological condition. The microrobot backbone was built with a biocompatible and elastic material-polydimethylsiloxane (PDMS)-by using a specially designed 3D molding aligner, and consisted of three strips of PDMS "legs" connected across a "body." Cardiomyocytes were then plated on the grooved top surface of the backbone, resulting in a high concentration of pulsating cells. These key techniques enabled the microrobot to walk continuously for over ten days. The performance of our crab-like microrobot was measured at an average velocity of 100 microm s(-1), and the estimated total distance it travelled was 50 m over a one-week period. Thus, we have demonstrated for the first time a walking robot that exhibited reliable and long-term actuation performances.

A three-dimensional model of fluid-structural interactions for quantifying the contractile force for cardiomyocytes on hybrid biopolymer microcantilever.

Quantitatively analysis of the contractility of cardiomyocytes is important for understanding the mechanism of heart failure as well as the molecular alterations in diseased heart cells. This paper presents a realistic computational model, which considers the three-dimensional fluid-structural interactions (FSI), to quantify the contractile force of cardiomyocytes on hybrid biopolymer microcantilevers. Prior to this study, only static modeling of the microscale cellular force has been reported. This study modeled the dynamics of cardiomyocytes on microcantilevers in a medium using the FSI. This realistic model was compared with static finite element modeling (FEM) analysis and the Stoney's equation-based analytical solution, and was validated by the deflections of the microcantilevers in the experimental results. Using harmonic response analysis in FSI modeling, the motion of a hybrid biopolymer microcantilever in the medium was identified as a second-order system and the influence of the dynamics of cardiomyocytes could be evaluated quantitatively.

Novel platform for minimizing cell loss on separation process: Droplet-based magnetically activated cell separator.

To reduce the problem of cell loss due to adhesion, one of the basic phenomena in microchannel, we proposed the droplet-based magnetically activated cell separator (DMACS). Based on the platform of the DMACS-which consists of permanent magnets, a coverslip with a circle-shaped boundary, and an injection tube-we could collect magnetically (CD45)-labeled (positive) cells with high purity and minimize cell loss due to adhesion. To compare separation efficiency between the MACS and the DMACS, the total number of cells before and after separation with both the separators was counted by flow cytometry. We could find that the number (3241/59 940) of cells lost in the DMACS is much less than that (22 360/59 940) in the MACS while the efficiency of cell separation in the DMACS (96.07%) is almost the same as that in the MACS (96.72%). Practically, with fluorescent images, it was visually confirmed that the statistical data are reliable. From the viability test by using Hoechst 33 342, it was also demonstrated that there was no cell damage on a gas-liquid interface. Conclusively, DMACS will be a powerful tool to separate rare cells and applicable as a separator, key component of lab-on-a-chip.

Evaluation of the critical stroke of an earthworm-like robot for capsule endoscopes.

Recently, the capsule endoscope has been highlighted for the patient's convenience and the possibility of application in the small intestine. However, the capsule endoscope has some limitations in obtaining an image of the digestive organ because its movement depends only on the peristaltic motion. In order to solve these problems, it is necessary to determine the locomotive mechanism of the capsule endoscope. Therefore, the present authors have already proposed an earthworm-like robot, which has a locomotive mechanism. However, this mechanism should be designed so that the earthworm-like robot has a larger stroke than the critical stroke required to perform motion inside the small intestine. In this study, therefore, not only is the modelling of the locomotive process based on a biomechanical study presented but also the movement of the earthworm-like robot in the small intestine is simulated. Through the simulation process, the variation in the critical stroke with regard to the elastic modulus of the mesentery is investigated. Finally, from an in vitro test of the proposed robot, it is found that the experimental result is very similar to that of the simulation. Consequently, the present work will provide guidelines for designing an earthworm-like robot for diagnosis of the small intestine.

Mechanical analysis of chorion softening in prehatching stages of zebrafish embryos.

During early development, the chorion envelope of the zebrafish embryo undergoes a thinning process called "chorion softening," which has so far only been characterized chemically. In this study, a micromechanical force sensing system was used to characterize and quantitate mechanical modifications of the zebrafish embryo chorion during early development. Quantitative relationships between applied forces and chorion structural deformations were established at various embryonic stages. The measured penetration force into the chorion at the blastula stage was 1.3-fold greater than those at the prehatching stage. Furthermore, chorion elastic modulus values were determined by using a biomembrane elastic model. The elastic modulus of the chorion at the blastula stage was 1.66-fold greater than that at the prehatching stage, thus indicating that the chorion envelope become mechanically "softened" at the prehatching stage. The experimental results quantitatively describe "chorion softening," which is most likely due to proteolytic activities at the prehatching stage. Gradual chorion softening during embryonic development was also artificially achieved by treating blastula chorion with pronase, a proteolytic enzyme. The forces required to penetrate the pronase-treated chorion were similar to those at the prehatching stage. This similarity suggests that "chorion softening" may be induced by the release of protease from the embryos, and the chemical nature of the process involves proteolytic fragmentation of the ZP2 protein.

An efficient cell separation system using 3D-asymmetric microelectrodes.

An efficient 3D-asymmetric microelectrode system for high-throughput was designed and fabricated to enhance sorting sensitivities to the dielectric properties-size, morphology, conductivity, and permittivity-of living cells. The principle of the present system is based on the use of the relative strengths of negative dielectrophoretic and drag forces, as in a conventional 3D-microelectrode system. Whereas the typical 3D-microelectrode system has a constant electric field magnitude due to the constant width of the microelectrodes and a fixed gap between face-to-face microelectrodes, the present 3D-asymmetric microelectrode system has electric fields of continuously varying magnitudes along the transverse direction of a channel owing to the changing widths of the electrodes in the half-circular shaped cross section of the microchannel. Thus, varying dielectric forces are generated, leading to increased sorting sensitivity through differentially induced forces to definitely distinct cell types. Numerical analysis verified the improved sensitivity of the present system for sorting living cells. The feasibility of using the newly fabricated system under experimental conditions was tested by demonstrating that a mixed population of mouse P19 embryonic carcinoma (EC) and red blood cells (RBCs) was effectively sorted to different wells depending on their respective relative physical properties.

Design and fabrication of an integrated cell processor for single embryo cell manipulation.

This paper presents an integrated cell processor for the automatic handling of individual embryo cells. The integrated processor can perform various functions such as cell transport, isolation, orientation, and immobilization. These functions are indispensable and frequently used for the manipulation of single cells, but can only be carried out by a skillful operator. The purpose of this study was the integration and automation of these functions for effective cell manipulation, using a MEMS approach. The isolation of a cell was performed using polypyrrole (PPy) valves in a microchannel into which cells were transported. The orientation of cells was controlled by electrorotation (ER), and the target cell was immobilized by suction from a microhole. All of these functions were seamlessly realized on a single chip. Excellent experimental results with mouse (B6CBA) embryo cells showed that this device could substitute for routine and cumbersome manual work. It is expected that the integrated chip will contribute significantly to faster and more reliable manipulation of cells.

Design and fabrication of a largely deformable sensorized polymer actuator.

Polypyrrole (PPy), with its biomimetic properties such as high power density, large strain, and biocompatibility, is an excellent candidate for a biomimetic microactuator in microrobotics and bioengineering. A polyvinylidene fluorid (PVDF) sensor is also biocompatible, flexible, and chemically stable. Therefore, a PPy actuator is integrated with a PVDF sensor to realize a sensorized polymer actuator. A novel sensorized polymer actuator can accurately measure its bending motion precisely with real time. Experimental results demonstrate the feasibility of the sensorized polymer actuator. The polymer actuator can be actuated while it senses signals induced from the bending motion. In addition, the position of the sensorized polymer actuator can be controlled and adjusted precisely with feedback signals from its embedded sensor at the time of operation. If this system becomes more robust and reliable, its applications are promising and can be realized in cell handling, microrobotics, and microsurgery with the integration of standard microfabrication techniques.

Investigating chorion softening of zebrafish embryos with a microrobotic force sensing system.

The zebrafish is a model organism for addressing questions of vertebrate embryo development. In this paper, the softening phenomenon of the chorion envelope of zebrafish embryos at different developmental stages was mechanically quantitated by using a microrobotic force sensing system. The microrobotic system integrates a piezoelectric cellular force sensor to measure the required forces for penetrating the chorion envelope. Magnitude of penetration forces was found to decrease as an embryo develops. The results mechanically quantitate "chorion softening" in zebrafish embryos due to protease activities subtly modifying the chorion structure, providing an understanding of zebrafish embryo development.

A negative dielectrophoresis and gravity-driven flow-based high-throughput and high-efficiency cell-sorting system.

We present a negative dielectrophoresis (n-DEP)-based cell separation system for high-throughput and high-efficiency cell separation. To achieve a high throughput, the proposed system comprises macro-sized channel and cantilever-type electrode (CE) arrays (L × W × H = 150 µm × 500 µm × 50 µm) to generate n-DEP force. For high efficiency, double separation modules, which have macro-sized channels and CE arrays in each separation module, are employed. In addition, flow regulators to precisely control the hydrodynamic force are allocated for each outlet. Because the hydrodynamic force and the n-DEP force acting on the target cell are the main determinants of the separation efficiency, we evaluate the theoretical amount of hydrodynamic force and n-DEP force acting on each target cell. Based on theoretical results, separation conditions are experimentally investigated. Finally, to demonstrate the separation performance, we performed the separation of target cells (live K562) from nontarget cells (dead K562) under conditions of low voltage (7Vp-p with 100 kHz) and a flow rate of 15 µL•min⁻¹, 6 µL•min⁻¹, and 8 µL•min⁻¹ in outlets 1, 2, and 3, respectively. The system can separate target cells with 95% separation efficiency in the case of the ratio of 5:1 (live K562:dead K562).

Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells.

The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show, consistent with previous studies conducted in other types of epithelial cancer, that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed examination of highly invasive ovarian cancer cells (HEY A8) relative to their less invasive parental cells (HEY), demonstrates that deformability is also an accurate biomarker of metastatic potential. Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. Our results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.