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    Neural cell differentiation

    Neural cell differentiation
    HCS confronts the challenges for high-throughput
    and secondary screening assays.
    BY PAUL WYLIE
    Control 50 µM ATRA
    TTP LABTECH
    SHSY-5Y differentiation. Acumen Explorer images of 4-Di-1-ASP-stained control
    and 7-day ATRA-stimulated cells (ATRA is all-trans-retinoic acid). Stimulated
    cells display the distinct formation of interconnecting neurite outgrowths.
    62 MODERN DRUG DISCOVERY JULY 2004
    Acumen Explorer, a laser-scanning fluorescence
    microplate cytometer, can rapidly
    detect and quantify all fluorescent objects
    in 96- to 1536-well formats and beyond.
    The Acumen Explorer differs from fluorescence
    microscopy systems in that it
    does not use microscope objectives or autofocusing
    optics during scanning. Because
    of focus-free, area-based scanning, the system
    permits multiplexed, whole-well HCS
    analysis with exceptionally fast read times,
    down to 3 min/plate, which is compatible
    with primary screening timelines. The algorithms
    use thresholding to identify fluorescent
    objects in a well, resulting in much
    smaller file sizes than those obtained by fluorescence
    microscopy systems.
    The role of HCS in drug screening programs
    has grown rapidly over the last 3-4
    years, primarily because of improvements
    in instrumentation and associated software.
    Other major advances include the
    application of fluorescent biomolecules,
    such as green fluorescent protein, which
    are used as intracellular protein markers.
    Labeled antibodies can also be extensively
    used to determine end-point analyses.
    Finally, there are growing numbers of fluorescently
    labeled dyes that stain a plethora
    of cellular structures, all of which can be
    used for cellular analysis. These advances
    let researchers analyze multicolor fluorescence
    and hence screen for multiple
    cellular readouts using a single assay in
    either fixed or live cells.
    Neuritogenesis assays
    Traditionally, assays for neurite outgrowth
    have used subjective, labor-intensive methods
    such as manually counting cells using
    fluorescence or confocal microscopy, but
    more recently, automated methods have
    become available. Automated monitoring
    and quantification of neurite formation and
    outgrowth in multiple samples have greatly
    enhanced therapeutic investigations in the
    neurobiological sciences, particularly in
    drug screening and the drug discovery
    process in general (3). However, these
    methods still offer relatively low throughput
    in terms of detection when compared
    with other higher-throughput screens, and
    they are limited to targeted screens or
    purely secondary screens. Another disadvantage
    is that these methods, and the
    instrumentation used, often require specialist
    cell lines-for example, Neuroscreen-
    1 cells for the ArrayScan screening assay-
    specific vendor-supplied algorithms, and the
    use of sometimes costly antibodies directed
    against proteins found in neurites.
    The ArrayScan assay utilizes PC12 cells,
    a rat pheochromocytoma cell line widely
    used as a standard model system for neurons
    (4). The assay identifies neurites
    using a primary antibody directed against
    tubulin and an Alexa Fluor 488 secondary
    antibody. An algorithm is applied to analyze
    the images with an option of applying
    Neuroscreen-1 cells, which are a subclone
    of the PC12 cells, for higher-throughput
    screening. The primary advantages of using
    these cells is that they grow 50-80% faster
    than wild-type PC12 cells and have a high
    and accelerated responsiveness to nerve
    growth factor (measurable neurites appear
    within 2 days rather than 6-8 days), making
    them more amenable to higherthroughput
    screening assays.
    The Discovery-1 system uses embryonic
    mouse day 13.5 trigeminal neurons.
    The neurons are labeled using anti-PGP9.5,
    followed by Alexa Fluor 488 secondary
    antibody. Like the ArrayScan assay, vendorsupplied
    algorithms are required to analyze
    the images and determine changes in cell
    morphology, including straightness and
    number of branch points.
    Finally, the Acumen Explorer-based assay
    measures neuronal cell differentiation of SHSY5Y
    cells, a human neuroblastoma cell line,
    by the incorporation of a membrane dye
    (see figure). The major advantage of using
    a human cell line is the potential to provide
    data that is more relevant to the likely therapeutic
    effects of compounds when applied
    to human biology. Unlike other assays, the
    Acumen Explorer uses live cells rather
    than fixed cells, allowing changes to be
    tracked over several days. The assay does
    not measure the primary morphological
    change in neurite formation, but, instead,
    it utilizes changes in dye intensity as a secondary
    indicator of cell differentiation.
    Because of this method of analysis, the
    Acumen Explorer assay is not as precise as
    microscope-based systems. However, the
    assay can rapidly analyze 96- or 384-well
    plates with plate read times on the order
    of 8 min/plate, thereby making it possible
    to run primary screens. Additionally, the
    open architecture of the Acumen Explorer
    software offers the potential for creating
    user-specific assays, for example, by changing
    cell lines or dyes, without having to purchase
    new algorithms.
    Rapid and slow HCS
    The Acumen Explorer offers a rapid, primary
    screening solution to neuronal cell differentiation
    with the potential to run up to
    20,000 compounds/day. However, the assay
    gives a less precise determination of neuronal
    cell differentiation, in contrast to the
    microscope-based HCS systems, but the
    Acumen Explorer is able to identify hits
    from a large compound library primary
    screen. As stated above, for neuritogenesis
    assays, different technologies are generally
    suited to either primary or secondary
    screens, and in general, two major types of
    instrumentation have been developed for
    HCS, fluorescence microscopy and laserscanning
    cytometry. The possible role that
    both these technologies could play in these
    methodologies has been discussed in this
    article. Because of their slower read times-
    it routinely takes in excess of an hour to read
    and analyze a 96-well plate-they are ideally
    placed to run secondary screens, where
    more time is available to gain a greater level
    of information, rather than primary screening
    on smaller targeted libraries. Together,
    these technologies may form a complementary
    combination to allow neuritogenesis
    screens in a high-content system.
    Therefore, through a combination of
    currently available HCS technologies, it is
    possible to run a fast primary screen and
    a slower secondary neuritogenesis screen
    using a high-content format in whole cells
    to determine morphological changes in
    response to drugs.
    References
    (1) Garyantes, T.; et al. A retrospective analysis of
    HCS-enabled lead discovery. Oral presentation
    at Drug Discovery and Technology 2003, Boston,
    Aug 10-15, 2003.
    (2) Alton, G. R.; Westwick, J. K. Eur. Pharm. Rev.
    2003, 3, 41.
    (3) Simpson, P. B.; et al. Anal. Biochem. 2001, 298,
    163-169.
    (4) Nakashima, S.; et al. Biochem. J. 2003, 376,
    655-666.
    Paul Wylie is a senior scientist at TTP LabTech
    Jake's Pop
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