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Cytotoxicity tests for high-throughput drug discovery

Kevin Slater
Despite theoretical obstacles associated with performing cellbased assays in high-density formats (microplates with at least
384 wells), it is becoming clear that the pharmaceutical
industry is now routinely running cell-based tests in these
formats. This work is revealing the weakness of established
cytotoxicity end points, specifically in relation to sensitivity and
reproducibility. New assay kits based on bioluminescent
detection of ATP and ADP are now providing the answer to
these problems.
Addresses
LumiTech Ltd, Nottingham Business Park, City Link, Nottingham,
NG2 4LA, UK; e-mail: k.slater@lumitech.co.uk
Current Opinion in Biotechnology 2001, 12:7074
0958-1669/01/$ see front matter
2001 Elsevier Science Ltd. All rights reserved.
Abbreviation
HTS
high-throughput screening

Introduction
This review will discuss the challenges facing the pharmaceutical industry resulting from the vast increases in
sample numbers produced by high-throughput screening
(HTS). Specifically, it will focus on the bottle necks created by increased demand for cytotoxicity testing (required
to assess compound safety) and will address the rapid
methods that are being developed to overcome this growing problem. Much of the information was obtained from
the Society for Biomolecular Screening (SBS) annual conference in Vancouver (69 September 2000). Otherwise
the reviewed articles cover the year between October 1999
and October 2000. Occasionally, I have made reference to
older papers in order to illustrate a point where necessary.

The ever-growing need for rapid cytotoxicity

assays in drug discovery
Todays pharmaceutical industry is faced with the unprecedented challenge of managing the progress of a rapidly
expanding pool of molecular targets, novel compounds and
biological assays, which are needed to discover and develop new drugs. Factors such as the near completion of the
Human Genome Project (producing an ever growing number of novel drug targets), combinatorial chemistry and
ongoing mergers between pharmaceutical companies add
to the scale and complexity associated with drug discovery.
Despite this, as well as the fact that HTS only began in the
1990s, compounds with therapeutic potential are already
emerging from combinatorial libraries [1].
During the past decade, the speed of screening has
increased dramatically. For example, using technologies
available in 1992 it would have taken the equivalent of 20
years to screen 1 million compounds. By 1996, the same

number of compounds could have been screened in 200

weeks. Using screening systems now in place in the pharmaceutical industry this has been reduced to only two
weeks. This phenomenal progress has been achieved
through assay miniaturisation, parallel processing and
innovations in hardware and assay technologies. It is now
common practice to run primary screens (e.g. receptorligand binding assays, proteinprotein interactions and
enzyme assays) comprising 150 384-well microplates per
day using modern HTS robots. The most common detection systems in HTS are fluorescence, scintillation
proximity assays (SPA) and luminescence. All these techniques provide excellent sensitivity (to facilitate
miniaturisation) and enable ultrahigh-speed measurements (using luminescence or fluorescence imaging
devices, such as the P.E. Lifesciences ViewLux or the
Molecular Devices CLIPR, an entire 384-well microplate
can be read in one second).
As a compound, identified in primary HTS (a hit), moves
further down the drug-discovery pipeline the costs associated with its failure increase dramatically. Considerable
effort is therefore given to ensuring that only the highest
quality compounds progress to further testing. Primary
screening will result in only 12% of the initial compounds
becoming hits. Secondary screening is then employed to
further reduce the number of hits to produce leads.
Functional assays based on whole cells, such as luciferase
reporter gene assays, are routinely employed as secondary
screens [2]. The objective of primary and secondary
screening is to reduce the number of lead compounds that
progress through the drug-discovery pipeline to those that
show true potential of becoming genuine product candidates. The number of leads now emerging from HTS
laboratories is, however, producing a new bottleneck in
drug discovery specifically, that of toxicity testing.
Pharmaceutical companies spend more money on compounds that subsequently fail because of their toxicity than
any other area. Toxicity testing was traditionally performed
prior to Phase 1 clinical trials where the costs of being wrong
can be immense. Scientists involved in ADME (absorption,
distribution, metabolism and excretion) and toxicology are
consequently re-evaluating their strategies, resulting in the
need for early phase toxicity testing. Traditional, animalbased methods are too slow, labour intensive and far too
costly for the present day demands of lead optimisation.
The role of in vitro cytotoxicity testing is therefore being
seriously evaluated and developed by the pharmaceutical
industry. Automation for HTS places new demands on cellbased assays. Issues such as the provision of the appropriate
cells in adequate numbers and the speed of cytotoxicity
tests are now paramount. Furthermore, parameters such as
signal:background ratio, reproducibility, simplicity and cost

Cytotoxicity tests for high-throughput drug discovery Slater

all take on a new significance and complexity when applied

to HTS. This article will attempt to address these issues in
relation to establishing high throughput assays of cytotoxicity suitable for drug discovery.

High-throughput cell-based assays a problem

of cell supply
There is a clear move within the pharmaceutical industry
towards increased emphasis on cell-based assays. This parallels the need for functional screens to discover what
happens within a cell when a molecule interacts with its target. This drive towards cell-based screening demands not
only large quantities of an individual cell type, it also
requires parallel processing of multiple, smaller batches of
a wide range of different cell lines derived from various tissue origins. Scale and flexibility are therefore demanded by
the pharmaceutical industry. Having large quantities of different cell types available on demand requires continuous
maintenance of many live cell cultures. The alternative to
maintaining and resuscitating frozen stocks is often viewed
as unacceptable because it can introduce unforeseen delays
in scheduling assays on laboratory robots. Two approaches
have been adopted for this problem: firstly, supply of
screening quantities of pre-plated cells from commercial
organisations; And secondly, the use of cell culture robots,
which incorporate incubators, laminar air flow and an
anthropomorphic arm, which is capable of seeding, feeding,
trypsinizing, harvesting and counting cells and dispensing
them into microplates for subsequent analysis.
The importance of this aspect of cell-based screening is
illustrated by the formation of a consortium of six major
pharmaceutical companies with a cell culture robot manufacturer with the objective of having automated bulk
cell-culture systems in operation by fourth quarter 2001 [3].
The high cost of obtaining cells for HTS is fuelling the drive
towards high content screening (HCS) that facilitates multiple
analyses on cell samples. HCS involves labelling individual
cell constituents with green fluorescent protein (GFP) variants
emitting at different wavelengths. Imaging systems are then
used to study the translocation within the cell of the labelled
targets, thus providing detailed information about the temporal and spatial dynamics of cell processes and components. By
imaging each cell individually, it becomes possible to treat
cells as wells within a 96-well microplate [4].

Cytotoxicity assays suitable for HTS

For the various reasons outlined above, toxicologists in the
pharmaceutical industry are now looking for new biomarkers of toxicity that will lead to high-throughput preclinical
safety assessment. A variety of different cell lines have
been used to predict organ specific toxicity; however, target organ screening in a high-throughput mode is an area
that requires further development [5].
The parameters for a successful HTS assay are now well
established. Assays should facilitate miniaturization to 96-,

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384- or 1536-well formats without detrimental effects on

robustness, reproducibility and statistical significance of
the results. To function within these parameters on laboratory robots, homogeneous mix and measure assays are
required as they avoid filtration, separation and washing
steps that are time consuming and difficult to automate [6].
Sensitivity is paramount for early phase cytotoxicity assays.
Historically, a high proportion of late pipeline failures has
occurred because of the detection of toxicity at a time
when costs are reaching their maximum. A number of dyebased cytotoxicity assays that superficially appeared to
address the parameters required for HTS were subsequently found to be lacking as they missed cytotoxic
effects resulting in the worse-case scenario of the late
pipeline dropout (various personal communications).
Dye-based assay

Historically, in vitro cytotoxicity assays have relied on a

number of end points. Firstly, chemical dyes, such as crystal violet or sulphorhodamine B, which stain specific cell
components and measure residual cellular material following incubation with the test compound. Secondly, detection
of release of a constitutive cellular component, such as lactate dehydrogenase, and subsequent measurement of the
enzymes activity in culture supernatant. Finally, measurement of cellular metabolic function using tetrazolium salts
(MTT, MTS, XTT), which are reduced to intensely
coloured formazan dyes by mitochondrial activity.
With the exception of the newer tetrazolium salts MTS and
XTT, none of these assays can be considered to be homogeneous and therefore robot friendly, requiring as they do
pre-incubation and washing steps. Over recent years the
focus has tended towards either fluorescent or luminescent
endpoints, which facilitate much more simple and convenient assay procedures and considerably higher levels of
sensitivity can be achieved using light-emitting chemistries.
There are a wide range of different fluorescent dyes available for use in cell viability studies. Many of these dyes
rely on the observation that viable cells possess intact plasma membrane structures that exclude the dye from the
cell, whereas cells treated with cytotoxic agents exhibit
plasma membrane disruption, which facilitates ingress of
the dye to the cytosol. However, King [7], in a very
detailed review of fluorescent dyes used in the measurement of cell killing, points out that a number of exceptions
to this observation exist and care should be taken in interpreting data obtained using some of the fluorescent dyes
that are now available. For example, although a cell may be
mortally wounded by a drug, it may still retain membrane
integrity for a relatively long time. Under these circumstances, a dye exclusion method would miss critical
cytotoxic effects so feared in cytotoxicity screening. King
reviews the use of fluorescent dyes in flow cytometric
analysis, which is an excellent tool for studying changes to
subpopulations within a cell culture. He raises an interesting point by highlighting that healthy, surviving cells

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within a population may proliferate following drug treatment. He therefore stresses that it is the absolute number
of surviving cells that is the most important parameter
rather than the percentage of dead cells, which is often
determined by flow cytometry. This is an observation that
we have regularly noticed in our own laboratory.
Despite recent developments with high-speed machines [8],
flow cytometry is currently not suited to the demands of
high-throughput cytotoxicity testing. High-throughput cytotoxicity assays need to be performed in microplates where
incubation and analysis can be achieved in the same plate and
a number of fluorescent microplate assays have been developed. Wodnicka et al. [9] have used a ruthenium dye (tris
4,7-diphenyl-1, 10-phenanthroline ruthenium [II] chloride) to
act as a biosensor. The fluorescence produced by this dye is
substantially quenched in the presence of oxygen. Thus, as
cells grow, the oxygen present in the medium diminishes over
time. This can be detected by an increase in fluorescence
resulting from a reduction in the quenching effect. Since the
ruthenium was not toxic to the cell types tested (CHO,
CaCo2, Alma16, MDCK, HL60, U-937, SF9, Saccharomyces
cerevisiae and Escherichia coli), it was possible to monitor the
growth of cells during the culture period by measuring
increase in fluorescence with a microplate fluorimeter.
Addition of cytotoxic agents to the cultures reversed this
effect, thus providing a very simple and rapid microplate cytotoxicity assay. From the data presented, however, it appears
that sensitivity is rather low, requiring 50,000 HL60 cells to
achieve a significant signal. Cells with higher metabolic rates,
and therefore greater oxygen consumption, could be detected with greater sensitivity and the insect cell SF9 was cited as
an example of this. Although the dynamic range of the assay
appears rather narrow (sixfold compared to a number of logs
for other assays), the method does facilitate kinetic analysis of
toxic effects, which the authors suggest may provide predictive value to indicate a drugs mode of action.
Another fluorescent dye, Alamar Blue (resazurin), lends itself
well to the demands of high-throughput cytotoxicity testing.
Oxidized, blue non-fluorescent Alamar Blue is reduced to a
pink fluorescent dye in cell-culture medium as a result of cell
activity. The exact mechanism of this dye reduction remains
to be elucidated; however, it is thought to be induced by
mitchondrial enzymes [10]. The test is very simple to perform, requiring the addition of only one reagent to the cell
culture supernatant (therefore providing a homogeneous
assay). It does not kill the cells, thus facilitating additional
tests on the same sample (which can be very important when
valuable primary cells are used). Most importantly, Alamar
Blue can provide a high degree of sensitivity, detecting a few
as 80 cells [11]. Although Alamar Blue can be co-cultured
with the cells giving the opportunity for kinetic analysis of
cytotoxicity, OBrien et al. [11] noted that compounds such as
nickel when added to cultures resulted in the reduction of the
dye even though the cells were dead, thus overestimating cell
survival. He concluded that kinetic experiments may be
unreliable unless strictly controlled.

Bioluminescent assays

Underestimation of toxic effects using the Alamar Blue

assay has been reported elsewhere. Martin [12] compared
the performance of six different cytotoxicity end-point
assays with HepG2 cells in the presence of 12 different
toxic compounds exhibiting different mechanisms of toxicity. She found that the most sensitive and reliable method
for detecting cytotoxicity was bioluminescent detection of
adenosine triphosphate (ATP). These findings concur with
our own [13]. Bioluminescent detection of ATP using the
firefly luciferase enzyme provides a very simple, highly
reproducible and extremely sensitive assay. ATP plays a
central role in energy exchanges in biological systems
(both eukaryotic and prokaryotic). It serves as the principal
donor of free energy and is present in all metabolically
active cells. ATP has been used as a tool to assess the functional integrity of living cells because all cells require ATP
to remain alive. Cell injury and death result in the rapid
decrease in cytoplasmic ATP [14]. Using the bioluminescent assay of ATP, we have developed a method (the
ViaLight assay) that is capable of measuring cytotoxicity in
all wells of a 96-well microplate in only three minutes. The
assay requires the addition of only one reagent, is capable
of detecting 10 cells per well and can cover a dynamic
range of six orders of magnitude. As mentioned earlier,
reproducibility is a very important consideration when
assays are transferred to laboratory robots. The simplicity
and precision afforded by bioluminescent analysis provides
highly reproducible assays in terms of inter and intra assay
variation. Furthermore, the extreme sensitivity that bioluminescence offers means that undesirable cytotoxic effects
are not missed as illustrated by Martin [12]. The benefits
of ATP bioluminescence compared to the widely used
MTT assay are clearly demonstrated in a paper by Cree
and co-workers [15].
As the trend for miniaturisation in HTS continues, the 384well microplate is gaining increasing prevalence. Aside
from the theoretical difficulties of growing cells in the small
volumes demanded by 384-well (and higher density)
microplates (because of issues of gaseous exchange resulting from the reduced surface area:volume ratio), many
pharmaceutical companies are now routinely culturing cells
in this format. The bioluminescent cytotoxicity assay has
been successfully applied to 384-well plates using primary
cultures of human tumour biopsies (IA Cree, personal communication). In this particular application, which provides
critical information about the most appropriate chemotherapeutic drug to use for an individual patient, it is not the
speed of the assay, but the extreme sensitivity and reproducibility of the bioluminescent method that provides the
benefits. These parameters enable a large number of assays
to be performed on a small tumour biopsy, thus allowing
many different drugs and combinations to be tested against
the sample and so increase the chances of finding the most
effective regimen for the patient. This 384-well work illustrates, however, that the bioluminescent method is
applicable to high-density screening.

Cytotoxicity tests for high-throughput drug discovery Slater

Advanced cytotoxicity testing

Over recent years, the concept of cell death has changed
radically as a result of the widespread interest in programmed cell death or apoptosis. This has lead to an
emerging understanding of the intracellular processes that
lead to apoptosis and the orderly removal of unwanted
cells from the body, and how control mechanisms of apoptosis breakdown in diseases such as cancer (too little
apoptosis) and Alzheimers disease (excessive apoptosis).
The socio-economic importance of the degenerative diseases is fuelling considerable research effort into cell death
in both academia and the pharmaceutical industry.
Lockshin et al. [16] has produced an excellent review on
the current status of thinking on cell death.
Of great importance to the study of cell death is the differentiation between the two principle modes, namely,
apoptosis and necrosis. Apoptosis is an active process that is
accompanied by a series of distinct cellular and molecular
events that form an integral part of normal physiological
processes. These include reduction in cell size, condensation
of nuclear chromatin and activation of endogenous endonucleases and proteases, which induce intracellular digestion of
the cell. In vivo, apoptosis is followed by removal of the cell
by phagocytosis. Necrosis, on the other hand, is the passive
result of cellular injury leading to complete loss of cell
integrity and the release of cellular components into the surrounding environment. When necrosis occurs in vivo it
results in a wide range of undesirable side effects including
inflammatory responses, which can be fatal in the extreme. It
is therefore important to discriminate between necrosis and
apoptosis in order to learn how to modulate apoptosis in view
of its potential therapeutic use.
Flow cytometric analysis remains the method of choice for the
study of apoptosis and necrosis. The full power of flow cytometry for the analysis of the multifarious events that occur
during apoptosis is reviewed by Vermes et al. [17]. However, as
discussed earlier in this article, flow cytometry is currently not
an ideal tool for screening large sample numbers. Although not
in widespread use at the moment, a number of high-throughput apoptosis assays have been developed.
We have extended our work on bioluminescent cytotoxicity assays to develop a rapid apoptosis screening method
based on the measurement of adenylate nucleotide ratios
(ATP and ADP) [18]. A considerable body of work on
apoptosis has focused on the mitochondria and it is now
widely accepted that this organelle is fundamental to the
biochemistry of apoptosis. Various biochemical pathways
involved in the apoptotic cascade converge on the mitochondria resulting in changes to membrane potential and
the release of cytochrome c into the cytosol. Cytochrome c
then initiates the activation of caspases, which mediate
intracellular digestion. The central role of the mitochondria in apoptosis, combined with the widely reported
findings that apoptosis is an energy requiring process, lead
us to investigate if subtle changes in energy metabolism

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(as determined by the relative levels of intracellular ATP

and ADP) could be used as a convenient and rapid apoptosis assay. Our findings clearly indicated that the
ADP:ATP ratio did indeed correlate with established flow
cytometric methods used to study apoptosis. Furthermore,
we found that from the magnitude of the ratio we could
differentiate between apoptosis and necrosis in cell cultures, with necrotic cells producing ADP:ATP ratios
around 10 times higher than apoptotic cells. Currently, we
can use the ADP:ATP ratio method to analyse a full
96-well microplate in 20 minutes in a fully automated fashion using luminometers with reagent injection facilities.

Conclusions
The objective of high-throughput screening is to reduce
the number of lead compounds to those that show true
potential of becoming genuine product candidates. As
pharmaceutical companies strive to improve the cost effectiveness of their drug-discovery programmes, it is
becoming clear that a considerable amount of money is lost
on compounds that fail late in the process because of their
toxicity. Their response to this has been to move toxicity
testing to an early phase in drug development. This is placing new demands on cytotoxicity tests, requiring
miniaturisation to 384-well formats, homogeneous mix
and measure assays and improvements in sensitivity.
Many of the established cytotoxicity endpoints, such as
tetrazolium dyes and Alamar Blue, have been successfully
miniaturised and can provide high-throughput analysis;
however, these methods were subsequently found to be
wanting because of poor sensitivity. Bioluminescent analysis of ATP appears to offer the answer to the demands of
speed and simplicity, and provides the sensitivity needed
to screen out low-level toxicity.

References and recommended reading

Papers of particular interest, published within the annual period of review,
have been highlighted as:

of special interest
of outstanding interest
1.

McMillan K: Identification and pharmacology of potent, selective

inhibitors of inducible nitric oxide synthetase. Abstract page 129
of the SBS 6th Annual Conference, Screening in the New
Millennium: 2000 September 6; Vancouver, Canada. Society for
Biomolecular Screening.

2.

Rees S: Ligand screening of GPCRs: the advantages of functional

assays. Abstract page 145 of the SBS 6th Annual Conference,
Screening in the New Millennium: 2000 September 6; Vancouver,
Canada. Society for Biomolecular Screening.

3.

Offin P, Drake R: The SelecT Consortium. J Assoc Lab Automat

2000, 5:20-22.

4.

Kain S: Green fluorescent protein (GFP): applications in cell

based assays for drug discovery. Drug Discov Today 1999,
4:304-312.
This interesting paper looks at innovative new methods in cell-based assys.
5. Johnson D, Wolfgang G: Predicting human safety: screening and

computational approaches. Drug Discov Today 2000, 5:445-454.

This paper describes the extension of applications of in vitro assys in predicting the safety of new drug compounds.
6.

Sundberg S: High-throughput and ultra-high-throughput

screening: solution- and cell-based approaches. Curr Opin
Biotechnol 2000, 11:47-53.

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7.
King M: Detection of dead cells and measurement of cell killing by
flow cytometry. J Immunol Methods 2000, 243:155-166.
This paper provides a comprehensive discussion of the benefits and pitfalls
of a wide variety of fluorescent dyes used in cell viability testing.
8.

9.

Ashcroft R, Lopez P: Commercial high speed machines open

new opportunities in high throughput flow cytometry (HTFC).
J Immunol Methods 2000, 243:13-24.
Wodnicka M, Guarino R, Hemperly J, Timmins M, Stitt D, Bruce Pitner J:
Novel fluorescent technology platform for high throughput
cytotoxicity and proliferation assays. J Biomol Screening 2000,
5:141-152.

10. De Fries R, Mistuhashi M: Quantification of mitogen induced human

lymphocyte proliferation: comparison of Alamar Blue assay to
3H-thymidine incorporation assay. J Clin Lab Anal 1995, 9:89-95.
11. OBrien J, Wilson I, Orton T, Pognan F: Investigation of the alamar

blue (resazurin) fluorescent dye for the assessment of

mammalian cell cytotoxicity. Eur J Biochem 2000, 267:5421-5426.
This paper highlights some of the pitfalls that can be encountered using
Alamar Blue in cell viability studies.
12. Martin T: High-throughput toxicity screening for cellular assays at the
re-test stage. Abstract of the SBS 5th Annual Conference: 1999
September 1316; Edinburgh, UK. Society for Biomolecular Screening.

13. Crouch SPM: The use of bioluminescent-based assays for cell

viability and apoptosis suitable for high-throughput screening
applications. Abstract page 95 of the SBS 6th Annual Conference,
Screening in the New Millennium: 2000 September 6; Vancouver,
Canada. Society for Biomolecular Screening.
14. Crouch S, Kozlowski R, Slater K, Fletcher J: The use of ATP
bioluminescence as a measure of proliferation and cytotoxicity.
J Immunol Methods 1993, 160:81-88.
15. Petty R, Sutherland L, Hunter E, Cree I: Comparison of MTT and
ATP-based assays for the measurement of viable cell number.
J Biolumin Chemilumin 1995, 10:29-34.
16. Lockshin R, Osborne B, Zakeri Z: Cell death in the third millennium.
Cell Death Differ 2000, 7:2-7.
This paper provides a clear description of intracellular mechanisms involved
in the processes of programmed cell deaths.
17.

Vermes I, Haanen C, Reutelingsperger C: Flow cytometry of

apoptotic cell death. J Immunol Methods 2000, 243:167-190.

18. Bradbury D, Simmons T, Slater K, Crouch S: Measurement of the

ADP:ATP ratio in human leukaemic cell lines can be used as an
indicator of cell viability, necrosis and apoptosis. J Immunol
Methods 2000, 240:79-92.
This paper provides a validation of the use of ATP/ADP ratios in apoptosis.