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