Jin Akagi

Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the real-time analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Wormometry-on-a-chip: Innovative technologies for in situ analysis of small multicellular organisms

Small multicellular organisms such as nematodes, fruit flies, clawed frogs, and zebrafish are emerging models for an increasing number of biomedical and environmental studies. They offer substantial advantages over cell lines and isolated tissues, providing analysis under normal physiological milieu of the whole organism. Many bioassays performed on these alternative animal models mirror with a high level of accuracy those performed on inherently low‐throughput, costly, and ethically controversial mammalian models of human disease. Analysis of small model organisms in a high‐throughput and high‐content manner is, however, still a challenging task not easily susceptible to laboratory automation. In this context, recent advances in photonics, electronics, as well as material sciences have facilitated the emergence of miniaturized bioanalytical systems collectively known as Lab‐on‐a‐Chip (LOC). These technologies combine micro‐ and nanoscale sciences, allowing the application of laminar fluid flow at ultralow volumes in spatially confined chip‐based circuitry. LOC technologies are particularly advantageous for the development of a wide array of automated functionalities. The present work outlines the development of innovative miniaturized chip‐based devices for the in situ analysis of small model organisms. We also introduce a new term “wormometry” to collectively distinguish these up‐and‐coming chip‐based technologies that go far beyond the conventional meaning of the term “cytometry.”

Real-time cell viability assays using a new anthracycline derivative DRAQ7®.

The exclusion of charged fluorescent dyes by intact cells has become a well‐established assay for determining viability of cells. In search for a noninvasive fluorescent probe capable of long‐term monitoring of cell death in real‐time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be nontoxic to a panel of cancer cell lines grown continuously for up to 72 h and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. The DRAQ7 provided a sensitive, real‐time readout of cell death induced by a variety of stressors such as hypoxia, starvation, and drug‐induced cytotoxicity. The overall responses to anticancer agents and resulting pharmacological dose‐response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real‐time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low‐dosage, real‐time labeling procedure provides multiparameter and kinetic fingerprint of anticancer drug action.

New rationale for large metazoan embryo manipulations on chip-based devices

The lack of technologies that combine automated manipulation, sorting, as well as immobilization of single metazoan embryos remains the key obstacle to high-throughput organism-based ecotoxicological analysis and drug screening routines. Noticeably, the major obstacle hampering the automated trapping and arraying of millimetre-sized embryos on chip-based devices is their substantial size and mass, which lead to rapid gravitational-induced sedimentation and strong inertial forces. In this work, we present a comprehensive mechanistic and design rationale for manipulation and passive trapping of individual zebrafish embryos using only hydrodynamic forces. We provide evidence that by employing innovative design features, highly efficient hydrodynamic positioning of large embryos on a chip can be achieved. We also show how computational fluid dynamics-guided design and the Lagrangian particle tracking modeling can be used to optimize the chip performance. Importantly, we show that rapid prototyping and medium scale fabrication of miniaturized devices can be greatly accelerated by combining high-speed laser prototyping with replica moulding in poly(dimethylsiloxane) instead of conventional photolithography techniques. Our work establishes a new paradigm for chip-based manipulation of large multicellular organisms with diameters well above 1 mm and masses often exceeding 1 mg. Passive docking of large embryos is an attractive alternative to provide high level of automation while alleviating potentially deleterious effects associated with the use of active chip actuation. This greatly expands the capabilities of bioanalyses performed on small model organisms and offers numerous and currently inaccessible laboratory automation advantages.

Multiparameter Lab-on-a-Chip flow cytometry of the cellcycle

Multiparameter analysis of apoptosis in relation to cell cycle position is helpful in exploring mechanism of action of anticancer drugs that target specific molecular cogs of the cell cycle. This work demonstrates a new rationale for using microfluidic Lab-on-a-Chip flow cytometry (μFCM) with a simple 2D hydrodynamic focusing for the multiparameter analysis of apoptosis and DNA ploidy analysis in human hematopoietic cancer cells. The microfluidic system employs disposable microfluidic cartridges fabricated using injection moulding in optically transparent poly(methylmethacrylate). The dedicated and miniaturized electronic hardware interface enables up to six parameter detections using a combination of spatially separated solid-state 473 nm (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide evidence that the simple 2D flow focusing on a chip-based device is sufficient to measure cellular DNA content in both fixed and living tumor cells. The feasibility of using the μFCM system for multiparameter analysis of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨ(m) loss) in relation to DNA content is also demonstrated. The data shows that straightforward microfluidic chip designs are sufficient to acquire high quality biological data when combined with sophisticated electronic interfaces. They can be a viable alternative to conventional FCM for multiparameter detection of programmed cell death.

Interfacing Cell-Based Assays in Environmental Scanning Electron Microscopy Using Dielectrophoresis

Development of the dielectrophoretic (DEP) live cell trapping technology and its interfacing with the environmental scanning electron microscopy (ESEM) is described. DEP microelectrode arrays were fabricated on glass substrate using photolithography and lift-off. Chip-based arrays were applied for ESEM analysis of DEP-trapped human leukemic cells. This work provides proof-of-concept interfacing of the DEP cell retention and trapping technology with ESEM to provide a high-resolution analysis of individual nonadherent cells.

Toward embedded laboratory automation for smart lab-on-a-chip embryo arrays

Lab-on-a-Chip (LOC) biomicrofluidic technologies are rapidly emerging bioanalytical tools that can miniaturize and revolutionize in situ research on embryos of small vertebrate model organisms such as zebrafish (Danio rerio) and clawed African frog (Xenopus laevis). Despite considerable progress being made in fabrication techniques of chip-based devices, they usually still require excessive and manual actuation and data acquisition that significantly reduce throughput and introduce operator-related analytical bias. This work describes the development of a proof-of-concept embedded platform that integrates an innovative LOC zebrafish embryo array technology with an electronic interface to provide higher levels of laboratory automation for in situ biotests. The integrated platform was designed to perform automatic immobilization, culture and treatment of developing zebrafish embryos during fish embryo toxicity (FET) biotests. The system was equipped with a stepper motor driven stage, solenoid-actuated pinch valves, miniaturized peristaltic pumps as well as Peltier heating module. Furthermore, a Field Programmable Gate Array (FPGA) was used to implement an embedded hardware/software solution and interface to enable real-time control over embryo loading and immobilization; accurate microfluidic flow control; temperature stabilization and also automatic time-resolved image acquisition of developing zebrafish embryos. This work presents evidence that integration of embedded electronic interfaces with microfluidic chip-based technologies can bring the Lab-on-a-Chip a step closer to fully automated analytical systems.

Automated Bio Cybernetic System: A Lab-on-Chip case study

This paper presents a high level systematic design approach for a distinctive type of application, automated Bio Cybernetic Systems (BCS), which enable experiments to be performed autonomously on live organisms in a Lab-on-Chip platform. The system integrates micro-electro-mechanical, microfluidics and embedded computing technologies into a fully Automated Biochemical Laboratory (ABL) with real-time sensing and actuating capabilities and control of multiple parallel experiments on large number of live organisms to achieve high throughput screening process. The system comprises of multiple concurrent control subsystems, imaging subsystem, higher-level data acquisition and storage system. A system level design language SystemJ is used to model the ABL as a Globally Asynchronous, Locally Synchronous (GALS) system in software and a hardware prototype is successfully built based on the software model.

Multiparameter Analysis of Apoptosis Using Lab‐on‐a‐Chip Flow Cytometry

The age of microfluidic flow cytometry (µFCM) is fast becoming a reality. One of the most exciting applications of miniaturized chip‐based cytometers is multivariate analysis using sampling volumes as small as 10 µl while matching the multiparameter data collection of conventional flow cytometers. We outline several innovative protocols for analyzing caspase‐dependent cell death and cell cycle (DNA‐content) profile using a fully integrated microfluidic flow cytometry system, Fishman‐R. The first protocol describes the use of a new plasma membrane–permeability marker, DRAQ7, and the fluorogenic caspase substrate PhiPhiLux to track caspase activation during programmed cell death. Also outlined is the use of DRAQ7 fluorochrome in conjunction with the mitochondrial membrane potential–sensitive probe TMRM to track dissipation of inner mitochondrial cross‐membrane potential. Another protocol adds the ability to measure dissipation of mitochondrial inner membrane potential (using TMRM probe) in relation to the cell cycle profile (using DRAQ5 probe) in living leukemic cells. Finally, we describe the combined use of fluorogenic caspases substrate PhiPhiLux with DRAQ5 probe to measure caspase activation in relation to the cell cycle profile in living tumor cells. Curr. Protoc. Cytom. 66:9.42.1‐9.42.15.