Although animal experiments are indispensable for preclinical screening in the drug discovery process, various issues such as ethical considerations and species differences remain. To solve these ...issues, cell-based assays using human-derived cells have been actively pursued. However, it remains difficult to accurately predict drug efficacy, toxicity, and organs interactions, because cultivated cells often do not retain their original organ functions and morphologies in conventional in vitro cell culture systems. In the μTAS research field, which is a part of biochemical engineering, the technologies of organ-on-a-chip, based on microfluidic devices built using microfabrication, have been widely studied recently as a novel in vitro organ model. Since it is possible to physically and chemically mimic the in vitro environment by using microfluidic device technology, maintenance of cellular function and morphology, and replication of organ interactions can be realized using organ-on-a-chip devices. So far, functions of various organs and tissues, such as the lung, liver, kidney, and gut have been reproduced as in vitro models. Furthermore, a body-on-a-chip, integrating multi organ functions on a microfluidic device, has also been proposed for prediction of organ interactions. We herein provide a background of microfluidic systems, organ-on-a-chip, Body-on-a-chip technologies, and their challenges in the future.
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The oceans sustain the global environment and diverse ecosystems through a variety of biogeochemical processes and their complex interactions. In order to understand the dynamism of the local or ...global marine environments, multimodal combined observations must be carried out in situ. On the other hand, instrumentation of in situ measurement techniques enabling biological and/or biochemical combined observations is challenging in aquatic environments, including the ocean, because biochemical flow analyses require a more complex configuration than physicochemical electrode sensors. Despite this technical hurdle, in situ analyzers have been developed to measure the concentrations of seawater contents such as nutrients, trace metals, and biological components. These technologies have been used for cutting-edge ocean observations to elucidate the biogeochemical properties of water mass with a high spatiotemporal resolution. In this context, the contribution of lab-on-a-chip (LoC) technology toward the miniaturization and functional integration of in situ analyzers has been gaining momentum. Due to their mountability, in situ LoC technologies provide ideal instrumentation for underwater analyzers, especially for miniaturized underwater observation platforms. Consequently, the appropriate combination of reliable LoC and underwater technologies is essential to realize practical in situ LoC analyzers suitable for underwater environments, including the deep sea. Moreover, the development of fundamental LoC technologies for underwater analyzers, which operate stably in extreme environments, should also contribute to in situ measurements for public or industrial purposes in harsh environments as well as the exploration of the extraterrestrial frontier.
Analysis of the intracellular materials of a small number of cancer cells at the single-cell level is important to improve our understanding of cellular heterogeneity in rare cells. To analyze an ...extremely small number of cancer cells (less than hundreds of cells), an efficient system is required in order to analyze target cells with minimal sample loss. Here, we present a novel approach utilizing an advanced electroactive double-well array (EdWA) for on-chip analysis of a small number of cancer cells at the single-cell level with minimal loss of target cells. The EdWA consisted of cell-sized trap-wells for deterministic single-cell trapping using dielectrophoresis and high aspect ratio reaction-wells for confining the cell lysates extracted by lysing trapped single cells via electroporation. We demonstrated a highly efficient single-cell arraying (a cell capture efficiency of 96 ± 3%) by trapping diluted human prostate cancer cells (PC3 cells). On-chip single-cell analysis was performed by measuring the intracellular β-galactosidase (β-gal) activity after lysing the trapped single cells inside a tightly enclosed EdWA in the presence of a fluorogenic enzyme substrate. The PC3 cells showed large cell-to-cell variations in β-gal activity although they were cultured under the same conditions in a culture dish. This simple and effective system has great potential for high throughput single-cell analysis of rare cells.
Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and ...providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network. This mechanism is based on programming deactivation laws using dedicated saturable pathways. We demonstrate this approach through the conversion of a single-node homoeostatic network into a bistable and reversible switch. Furthermore, we prove its generality by adding new functions to the library of reported man-made molecular devices: a system with three addressable bits of memory, and the first DNA-encoded excitable circuit. Specific saturable deactivation pathways thus greatly enrich the functional capability of a given circuit topology.
Reaction networks displaying bistability provide a chemical mechanism for long-term memory storage in cells, as exemplified by many epigenetic switches. These biological systems are not only bistable ...but switchable, in the sense that they can be flipped from one state to the other by application of specific molecular stimuli. We have reproduced such functions through the rational assembly of dynamic reaction networks based on basic DNA biochemistry. Rather than rewiring genetic systems as synthetic biology does in vivo, our strategy consists of building simplified dynamic analogs in vitro, in an artificial, well-controlled milieu. We report successively a bistable system, a two-input switchable memory element, and a single-input push-push memory circuit. These results suggest that it is possible to build complex time-responsive molecular circuits by following a modular approach to the design of dynamic in vitro behaviors. Our approach thus provides an unmatched opportunity to study topology/function relationships within dynamic reaction networks.
Microfluidic devices provide a number of advantageous features for microscale biochemical systems for analysis and/or synthesis. A PDMS (polydimethylsiloxane) microchip, for instance, which has ...microchannels for electrophoretic separation can be easily fabricated through a molding process. Sealing of those channels does not need any elaborate bonding processes which are usually required for glass chips. We have been working on PDMS-based microfluidic devices for biomedical applications, where microreactors, microchips for capillary gel electrophoresis, and hydrophobic vent valves are successfully fabricated and operated. Fundamentals of PDMS-based microfluidic devices and their functions are described as well as the experimental results.
The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring ...of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.
In vitro spermatogenesis (IVS) using air-liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, ...only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.
Spatial Waves in Synthetic Biochemical Networks Padirac, Adrien; Fujii, Teruo; Estévez-Torres, André ...
Journal of the American Chemical Society,
10/2013, Letnik:
135, Številka:
39
Journal Article
Recenzirano
We report the experimental observation of traveling concentration waves and spirals in a chemical reaction network built from the bottom up. The mechanism of the network is an oscillator of the ...predator–prey type, and this is the first time that predator–prey waves have been observed in the laboratory. The molecular encoding of the nonequilibrium behavior relies on small DNA oligonucleotides that enforce the network connectivity and three purified enzymes that control the reactivity. Wave velocities in the range 80–400 μm min–1 were measured. A reaction–diffusion model in quantitative agreement with the experiments is proposed. Three fundamental parameters are easy to tune in nucleic acid reaction networks: the topology of the network, the rate constants of the individual reactions, and the diffusion coefficients of the individual species. For this reason, we expect such networks to bring unprecedented opportunities for assaying the principles of spatiotemporal order formation in chemistry.
This paper presents a micro-flow diagnostic technique, 'high-speed confocal micro-particle image velocimetry (PIV)', and its application to the internal flow measurement of a droplet passing through ...a microchannel. A confocal micro-PIV system has been successfully constructed wherein a high-speed confocal scanner is combined with the conventional micro-PIV technique. The confocal micro-PIV system enables us to obtain a sequence of sharp and high-contrast cross-sectional particle images at 2000 frames s(-1). This study investigates the confocal depth, which is a significant parameter to determine the out-of-plane measurement resolution in confocal micro-PIV. Using the present confocal micro-PIV system, we can measure velocity distributions of micro-flows in a 228 microm x 171 microm region with a confocal depth of 1.88 microm. We also propose a three-dimensional velocity measurement method based on the confocal micro-PIV and the equation of continuity. This method enables us to measure three velocity components in a three-dimensional domain of micro flows. The confocal micro-PIV system is applied to the internal flow measurement of a droplet. We have measured three-dimensional distributions of three-component velocities of a droplet traveling in a 100 microm (width) x 58 microm (depth) channel. A volumetric velocity distribution inside a droplet is obtained by the confocal micro-PIV and the three-dimensional flow structure inside the droplet is investigated. The measurement results suggest that a three-dimensional and complex circulating flow is formed inside the droplet.
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