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Cell Analysis
Real-Time Cell-Based Toxicology Testing Might Replace
Animal Testing for Product Release and Drug Safety
James O’Connell*, Yama Abassi, Biao Xi, Xiaobo Wang, and Xiao Xu
ACEA Biosciences, San Diego, CA, USA
*Corresponding author: [email protected]
methods. Botulinum toxin type A (BOTOX®) is unique
among medical therapies in that it is a biological product,
which means that it is derived from natural sources – in
this case, from the bacterium C. botulinum. When manufacturing biological products, testing is particularly critical
to ensure the consistent safety and efficacy of each batch
of product. The safety and efficacy of botulinum toxin type
A is assessed by using the LD50 test (Lethal Dose 50%).
In the case of botulinum toxin type A, mice are injected
with the active ingredient – a form of the same toxin that
causes botulism food poisoning – and experience differing levels of muscular paralysis. Those given a high or
powerful dose eventually die from suffocation after their
respiratory muscles become paralyzed [1]. In addition,
over the past 10 years Allergan, the botulinum toxin type
A manufacturer, has invested more than 40 million U.S.
­dollars in the development of alternative assays which
they hope will be able to replace animal-based assays in
the manufacture of the product.
The xCELLigence System, codeveloped by Roche
and ACEA Biosciences, was used to conduct in vitro
testing of selected microbial toxins (e.g., Clostridium
botulinum toxin type A and the closely related
C. difficile toxin) and in vitro cardiotoxicity testing
of drug candidates. Currently, laboratory animals are
required to be used in the release of pharmaceutical
products such as botulinum toxin type A and in the
prediction of cardio­toxicity of new drugs. Such tests
consume very large numbers of laboratory animals.
We believe that the xCELLigence System can
essentially replace the animals used in these types
of testing. Furthermore, as the xCELLigence System
becomes linked to other systems, such as the Roche
454 System and the Roche NimbleGen Arrays, we
expect that animal testing will be reduced significantly in pharmaceutical development in general.
Background
Currently, all pharmaceutical manufacturers are required
by the Food and Drug Administration (FDA) in the United
States and by other worldwide health regulatory agencies
to protect patients and consumers by assuring product­
safety and efficacy through animal testing and other
b
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The xCELLigence System
The xCELLigence System allows label-free dynamic monitoring of living cells. The core of the system is the integrated microelectronic sensor contained in each well of
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Control
10x diluted positive sample
1,000x diluted positive sample
1,000x diluted positive sample + Ab
1 ng/ml Toxin A
1 ng/ml Toxin A + Ab
10 pg/ml Toxin A
1.0 pg/ml Toxin A
0.1 pg/ml Toxin A
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Normalized cell index
Normalized cell index
a
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Control
Toxin A 30 ng/ml
Toxin B 30 ng/ml
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James O'Connell
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Figure 1: Cell-based assay for C. difficle toxin A and B testing on the xCELLigence System. (a) Cytotoxic effect of toxins A and B. The toxic
effects of both toxins A and B were tested using a cell-based assay on the xCELLigence System. Cytotoxic kinetic patterns are different for both toxins.
(b) Sensitivity and specificity of the toxin detection using fecal samples. The system is able to detect the toxin in fecal samples with great ­sensitivity
(1 pg/ml) and excellent specificity.
Biochemica · No. 4 · 2008
1212
Cell Analysis
the 96-well E-Plate. Application of a low-voltage AC current allows the microsensor to detect minor changes in
the ionic environment of the well, which are related to
changes in cell number, changes in cell morphology, and
the strength and quality of attachment of the cells to the
bottom surface of the microwell [2]. The system is ideally
suited for cell-based applications such as:
 Cellular quality control
 Cell proliferation
 Cytotoxicity
 Cell adhesion and spreading
 Receptor-mediated signaling
 Barrier function
xCELLigence Assays Designed
to Reduce Animal Testing in
Pharmaceutical Development
Microbial toxin assays
This cell-based assay on the xCELLigence System provides kinetic information which is not available with other
technologies.
Clostridium difficile is resistant to most antibiotics and
its ­toxins A and B cause colitis. The real-time monitoring
of the live cells using the xCELLigence System shows
the time-dependent cytotoxic effect of toxins A and B
and the unique cell death patterns associated with each
specific toxin (Figure 1a). This provides a great predictive
value for the in vitro cell-based assay.
In addition, we have used the xCELLigence System to
identify C. difficile toxins A and B [3] directly from stool
samples using cell culture and specific toxin neutralization with highly specific toxin A and B antibodies. The
a
1.5
fecal samples were obtained either from subjects infected
with C. difficle (positive samples) or from fecal samples
spiked with purified toxin A – which was added to the
cells as a control. The sensitivity was determined by the
fecal samples spiked with the serially diluted toxin. The
specificity was determined by the neutralization of the
toxic effect by specific antibody. The test as performed
with the ­xCELLigence System is extremely sensitive – in
the pg/ml range – and highly specific (Figure 1b).
We also were able to use the system to detect the biological
effect of botulinum toxin, which is approxiamtely 1 billion
U.S. dollar/year drug. Currently there is no in vitro assay
approved by the U.S. FDA (and other international regulatory bodies) for the release of botulinum toxin A. However,
using the xCELLigence System, we were able to detect the
effect of toxin on CNS cell lines A172 glioblastoma cell line
and SH-SY5Y neuroblastoma cell line. Cells were treated
with botulinum toxin at a concentration of 6.67 µg/ml, and
the effect was contiuously monitored on the xCELLigence
System (Figure 2). Since botulinum toxin can have a complex effect on the cells, including binding to cell surface
receptors, uptake, processing, and prevention of synaptic
vesicle anchoring to the cell membrane, we are currently
trying to understand how these processes contribute to the
cellular response detected by the xCELLigence System. We
believe the quantitative detection of botulinum toxin effect
on CNS cells by the xCELLigence System has the potential
of replacing the required animal test and further studies
are warranted to determine the extent of correlation with
LD50 data derived from animal testing.
Cardiotoxicity assays
Similarly, another important area that the xCELLigence
System could significantly impact is the cardiotoxicity
b
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Normalized cell index
Normalized cell index
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treatment
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Control
Botulinum toxin A
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Time (hours)
Figure 2: Dynamic monitoring of toxic effect of botulinum toxin on CNS cell lines using the xCELLigence
System. Botulinum toxin A at a concentration of 6.67 µg/ml was tested on (a) the A172 glioblastoma cell line
(ATCC) and (b) the SH-SY5Y neuroblastoma cell line (ATCC).
Biochemica · No. 4 · 2008
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Cell Analysis
Add compound
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10µM
5 µM
2.5 µM
1.25 µM
0.63 µM
0.31 µM
0.16 µM
DMSO
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Normalized cell index
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Log of concentration (M)
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-4.00
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c 6.0 x10
Figure 3: Monitoring of emetine-mediated
cardio­toxicity using mouse stem cell-derived
­cardiomyocytes (CorAT cells) on the xCELLigence
System. ­(a) Dose-dependent cytotoxic kinetics of
emetine. (b) Dose-dependent cardiotoxic effect at 24
hours of compound treatment. (c) Time-dependent
IC50 values during compound treatment. IC50 is a
quantitative measure indicating how much of a particular substance is needed to inhibit a given biological
process or component of a process (i.e., an enzyme,
cell, cell receptor or micro­organism) by half.
5.0 x10-7
IC50 (M)
4.0 x10-7
3.0 x10-7
2.0 x10-7
1.0 x10-7
0
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Time after drug addition (h)
assessment of drug candidates, which currently requires
the extensive use of laboratory animals. A major effort
has been underway to replace these tests with in vitro
tests, but the most substantial shortcoming of current
in vitro cardiotoxicity assessment methods is a lack of
­adequate predictivity [4]. The predictivity can be significantly improved by using a real-time, label-free cardiomyocyte-based assay on the xCELLigence System.
Based on our preliminary study with mouse stem cellderived cardiomyocytes, the real-time, continuous monitoring of cardiomyocytes in response to exposure to test
compounds allows for detection of early, transient ion
channel- or receptor-mediated effects and long-term
cardiomyo­cyto­toxicity in the same living cell population
(Figure 3). In our experiment, mouse stem cells were
seeded onto the 96-well E-plate and then differentiated
to cardiomyocytes (CorAT cells, Axiogenesis) in differentiation media. The differentiation was monitored in realtime. Once the stem cells had differentiated into specific
cardiomyocytes, the compound emetine was added in
different concentrations. The cardio­toxic effect was then
continuously monitored in real-time for an additional 24
hours (Figure 3).
In addition, with the unique features of the automatic
data acquisition and high-throughput assay format, the
xCELLigence System can be used in secondary screening­
as well, which makes the prediction and prioritization of
Biochemica · No. 4 · 2008
cardiotoxicity possible even in the early stage of drug discovery.
Conclusions
We believe that the combination of the xCELLligence
System with other high information content systems might
bring a new level of accuracy and information to in vitro
testing that will significantly reduce the number of animal
tests required in pharmaceutical development.
n
References
1.Humane Society of the United States statement on Botox® product
testing­ (www.hsus.org)
2. Abassi Y (2008) Biochemica 2:8–11
3. Yablon SA et al. (1992) Am J Phys Med Rehabil 71:102–107
4. Tomoaki I et al. (2007) Proc 6th World Congress on Alternatives &
Animal Use in the Life Sciences – AATEX 14 Special Issue 457–446
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