4 results for Akagi, JA

  • Metabolomics-on-a-chip: integrating microfluidic single cell culture with mass spectroscopy

    Wlodkowic, Donald; Wright, BEW; Akagi, JA; Greenwood, DG; Villas-Boas, SVB; Williams, DEW (2010)

    Conference item
    The University of Auckland Library

    We are developing microfluidic systems to study the metabolome of a single cell or small cell groups in well-defined culture conditions. [1] Microfluidic systems, also called labs-on-a-chip, are constructed on the same scale as cells (features in the range of 1-100 µm) cell culture numbers with high spatial and temporal control of culture conditions. [2] Toward this end, we have adapted the use of microfluidic single cell traps capable of capturing and housing the culture of a single cell or small cell groups using polydimethysiloxane (PDMS) based microfluidics. [3] Further, we have integrated microfluidic cell culture with novel monolithic PDMS electrospray ionisation (ESI) emitters. We have tested the fully integrated cell traps/ESI emitters using both test solutions and in trial runs on small yeast cell groups using an ion trap and fourier transform ion cyclotron resonance mass spectrometer. Initial results on the microfluidic cell traps, microfluidic-ESI characteristics, and performance of the integrated cell traps/ESI system will be presented along with an comparison of conventional nanospray-ESI with the integrate microfluidic system with emphasis placed on detection sensitivity and interference effects from PDMS.

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  • Dynamic Analysis of Tumour Cell Death Using Dielectrophoresis

    Wlodkowic, Donald; Khoshmanesh, KK; Akagi, JA; Nahavandi, SN; Skommer, JS; Baratchi, SB; Cooper, JMC; Kalantar-Zadeh, KKZ; Williams, DEW (2011)

    Conference item
    The University of Auckland Library

    The important discovery that induction of programmed cell death in neoplastic cells is a critical event that defines tumour growth rate and response to anti-cancer therapy has provided a framework for the rationally designed, molecular anti-cancer therapeutics. The regulation of cancer cell death involves the circuitry of feedback events and rapid switches between analogue and digital responses. Such enormous complexity, with numerous variables acting at the same time, requires multiparametric and dynamic analysis at a single cell level. The opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. We describe here the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilisation platform for the real-time analysis of tumour cell death. We applied this technology to the dynamic analysis of haematopoietic tumour 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 labelling with cell permeability markers. Results indicate that simple DEP cell immobilisation technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells.

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  • Real-Time Fluorescent Imaging of Apoptosis using Chip-Based Technologies

    Wlodkowic, Donald; Zhao, HZ; Akagi, JA; Dobrucki, JD; Williams, DEW; Cooper, JMC; Darzynkiewicz, ZD (2011)

    Conference item
    The University of Auckland Library

    Validation of potential therapeutic targets necessitates the development of new assays that provide both spatial and temporal relationships in signalling networks. Cell-based assays are therefore becoming an important part of the postgenomic biomedical research. The present study was designed to provide a mechanistic rationale for the kinetic assays and to assess their potential to obtain dynamic and single-cell data on the stochastic process of cell death on Lab-on-a-Chip (LOC) technologies. We propose an innovative approach to dynamically trace cell death in real-time using fluorochromes such as propidium iodide (PI), SYTOX Green, SYTOX Red, YO-PRO 1, Annexin V and TMRM. We also demonstrate that, when used with innovative bioassays, microfluidic dynamic live-cell analysis is a practical alternative for multiparameter studies on a single-cell level. The LOC devices not only reduce the complexity of conventional cell culture protocols but also enabled time-resolved studies on apoptosis. The adaptation of non-toxic biomarkers that can continuously circulate inside enclosed microculture system will be beneficial for the advancement of up and coming LOC technologies. Our data supports the hypothesis that real-time bioassays in combination with LOC devices allow for rapid and simple analysis of cell death, particularly useful if the death pattern is a stochastic rather than deterministic process. As a result, they provide sensitivity that often cannot be achieved with conventional end-point analysis.

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  • Interfacing dielectrophoretic cell trapping arrays with ESEM

    Wlodkowic, Donald; Khoshmanesh, KK; Akagi, JA; Nahavandi, SN; Kalantar-Zadeh, KKZ; Williams, DEW; Cooper, JMC (2011)

    Conference item
    The University of Auckland Library

    Live cell assays are an important part of drug discovery pipelines and personalized point-of-care diagnostics. In this context, Lab-on-a-Chip (LOC) devices are being widely considered as emerging technologies that can support massively parallel analysis at a single cell level. Reportedly they provide unique capabilities to monitor single-cell signalling dynamics, especially in rare subpopulations such as cancer cells or haematopoietic stem cells. Despite a large body of evidence on the fluorescence imaging, impendence and optical spectroscopies for characterisation of single living cells on a chip, however, no attempts have been so far made to interface microfabricated chip-based technologies with environmental scanning electron microscopy (ESEM). Here we for the first time describe the development of a non-invasive and simplified DEP chip that can be interfaced with ESEM imaging to provide analysis of non-adherent cells immobilised in the positive DEP (pDEP) fields. DEP microelectrode arrays were fabricated using thin films of chrome/gold deposited to a thickness of 500 Å/1500 Å on the glass substrate using electron beam evaporation process. Chip-based arrays were applied for ESEM analysis of DEP-immobilised human leukaemic cells. Most importantly non-adherent cells remain viable during processing and are well retained during imaging. Our innovative approach avoids extensive preparative procedures, is easy to perform for non-specialised personnel and thus prospectively amenable for automation.

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