Toxicology in Vitro 21 (2007) 176–182
www.elsevier.com/locate/toxinvit
Cytomics: A multiparametric, dynamic approach to cell research
Guadalupe Herrera, Laura Diaz, Alicia Martinez-Romero, Angela Gomes, Eva Villamón,
Robert C. Callaghan, José-Enrique O’Connor ¤
Laboratorio de Citómica, Unidad Mixta de Investigación CIPF-UVEG, Centro de Investigación Príncipe Felipe,
Avda. Autopista del Saler, 16, 46013 Valencia, Spain
Received 8 April 2006; accepted 13 July 2006
Available online 22 July 2006
Abstract
Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation
of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science
of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external
inXuences. Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based multiparametric methods and the event-integrating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies,
Xow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologies
based on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both highcontent and high-troughput. The multiparametric capacity of cytomics is very useful for the identiWcation, characterization and isolation
of stem cell populations. In our experience, Xow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic
assays leads to a real-time kinetic approach based on sequential examination of diVerent single cells from a population undergoing a
dynamic process, the in Xuxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Cytometry; Fluorescence; Toxicology; Pharmacology; In vitro
1. Cytomics and cytomes: single-cell based analysis
in complex systems
Genomics, proteomics and metabolomics provide outstanding technical contributions to Cell Biology but
become limited when single or scarce cells are examined or
fast cellular processes followed kinetically (Figeys, 2004). In
addition, the importance of epigenetic changes (Plass, 2002)
and the modulatory inXuence of cell environment (Abrous
et al., 2005) recommend to combine “-omic” studies with
functional analysis (Bernas et al., 2006). Because of this,
cell-based assays are increasingly sought for basic research
*
Corresponding author. Tel.: +34 963 28 9680; fax: +34 963 28 9671.
E-mail address: [email protected] (J.-E. O’Connor).
0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2006.07.003
and drug screening, since they approach complex cell-tocell and cell-to-environment interactions, in assay formats
that are both information-rich and technically convenient.
In this context, where “-omics” meet Systems Biology, cytomics represents a novel analytical strategy aimed to determine multiple biochemical features (the molecular
phenotype) in single cells and may be deWned as the cytometry of complex cellular systems.
In analogy with other -omics (genomics/genome; proteomics/proteome, and so on) the objective of cytomics are the
heterogeneous cellular systems known as the cytomes. The
cytomes can be understood as the heterogeneous cellular
systems and functional components of the pluricellular
organisms. Since the functional heterogeneity of the cytomes results from both the genome and extracellular environment, cytomics can be considered as a discipline that
G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
links genomics and proteomics to cell and tissue function,
as modulated by external inXuences. Of special importance
is the cell-by-cell basis of cytomic analysis, an approach
that allows to resolve heterogeneous systems and avoids the
loss of information that characterizes bulk technologies, in
which average values are obtained from large number of
cells or from tissue homogenates.
For a deeper, comprehensive view of cytomics and their
relevance to biomedicine, see Valet (2005) and Bernas et al.
(2006).
2. Technical and analytical features of cytomic technologies
Inherent to cytomics are the use of sensitive, scarcely
invasive, Xuorescence-based methods and the integrating
concept of individual cell analysis to understand the complexity and behaviour of tissues and organisms. Due to the
availability of large number of Xuorescent markers and the
multiplicity of Xuorescence detectors interfaced to the dedicated instrumentation, cytomic assays may be multiparametric, polychromatic and multiplexed. Fluorescence-based
measurements may be qualitative and quantitative and can
be obtained as the result of single end-point measurement
or kinetic, sequential measurements. While these features
are common to all cytomic technologies, there are important speciWc diVerences depending on whether the quantitative cell Xuorescence data are extracted together with cell
morphology in image-based cytomics (Eils and Athale,
2003) or from Xuorescence-pulse analysis in Xow-based
cytomics (O’Connor et al., 2001). Among the current
cytomic technologies, Xow cytometry (FCM), confocal
laser scanning microscopy (CLSM), spinning-disk confocal
microscopy (SDCM) and laser scanning cytometry (LSC)
are of established relevance. Other cytomic technologies
based on single-cell based image analysis and powerful bioinformatic tools (high-content screening bioimaging, HCSB) have been recently introduced for drug discovery and
toxicity testing, as they can provide both high-content and
high-troughput analysis. Finally, laser capture microdissection (LCM), a preparative technique for obtaining pure
cells from speciWc microscopic regions of tissue sections,
can also be considered among cytomic technologies.
177
susceptibility or resistance. The advantages of FCM derive
from its multiparametricity that provides multiple, simultaneous targets to assess cell lesion or death in selected cell
populations, either as end-point or kinetic measurements.
2.2. Confocal Xuorescence microscopy
Confocal Xuorescence microscopy is advantageous to
conventional Xuorescence microscopy, as it narrows the
Weld depth, eliminates out-of-focus blur and allows to produce serial optical stacks from thick specimens (z-axis resolution). Confocality may be applied to image single cells
from Wxed or living preparations labelled with appropriate
Xuorescent probes. This technique allows sophisticated cell
information based on spatial- and time-resolved Xuorescence measurements and as such is being increasingly used
for scientiWc and technological applications (Rubart, 2004;
Ivorra et al., 2006). Usually, CLSM yields better image
quality but the imaging frame rate is slow while SDCM
may produce video rate imaging, which is required for
eYcient dynamic observations (Maddox et al., 2003). However, the new confocal systems function like hybrids allowing to obtain both high speed and good resolution.
2.3. Laser scanning cytometry
LSC is a microscope-based, scanning cytoXuorimeter
that combines the advantages of Xow and image cytometry.
It allows multiparametric analysis performed directly by
measuring the Xuorescence of individual cells in solid-state
preparations, such as monolayers, smears, imprints, cytospins or tissue sections in several supports including slides,
dishes and multiwell plates. LSC provides increased sensitivity and speciWcity compared to traditional microscopic
techniques and a similar structure for data analysis to Xow
cytometry, albeit at lower data acquisition velocity. In addition, it allows relocation of the coordinates of analyzed
cells of interest following slide restaining. Finally, single
cells identiWed by their Xuorescence measurements can be
individualized from histogram or dot-plot displays and
shown as single-cell pictures or combined in picture galleries for further analysis (Juan and Cordon-Cardo, 2001).
2.1. Flow cytometry
2.4. High-content screening bioimaging
This methodology requires that cells (or microscopical
biological particles) are in suspension. FCM allows the
simultaneous quantiWcation of multiple Xuorescence emissions in the same cell, arising from Xuorescent markers, and
scattered light related to morphology, revealing key cellular
functions or structures (O’Connor et al., 2001). The velocity
of analysis can be up to thousands of single-cells per second
and individual cells from heterogeneous subpopulations
can be physically isolated on the basis of their Xuorescence
or light scatter properties. The multiparametric capacity of
FCM permits to quantify the eVects induced by the exposure to a toxic agent, providing a direct proof of cellular
An HCS-B system is typically composed by a motorized
Xuorescence microscope, a CCD camera that captures the
images, and a digitizing system that stores the images in a
large-capacity computer, with each step controlled by software for image acquisition and analysis. When applied to
high-troughput screening (HTS) cell-based assays this
novel technical concept allows cell research at large scale
using multiwell plates and analyzing in bulk millions of
cells per experiment. These systems may examine up to one
single well per second and combine both high-temporal and
spatial resolution. HCS-B is very eYcient for complex analyses that involve combinations of diVerent cell types and
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G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
concentrations of agonists and antagonists, varying times
of exposure as well as the type and number of parameters
studied on each condition. In addition, the possibility of
maintaining stable conditions of temperature and CO2
allows live cell functional assays. The detection in individual cells provides additional information on the responses
of heterogeneous subpopulations that may diVer in their
developmental stage, cell cycle phase, transfection status or
other parameters. However, individual cell analysis may
require special measurements (neurite outgrowth, nuclear
localization, etc.), that reduce the speed required to be qualiWed as both HTS and HCS (Abraham et al., 2004; Borchert
et al., 2005).
2.5. Laser capture microdissection
Meaningful proteomic and genomic analysis demand the
preparation of homogeneous cell populations. Flow-cytometry cell sorting may be used to purify a particular cell type
in suspension but not easily from solid tissue samples. To
improve cell isolation, several microdissection methods
have been developed, but are often time-consuming and
imprecise. LCM has been developed to automate and standardize microdissection, increasing reproducibility and
accuracy of selecting targeted cells from a complex tissue
for subsequent molecular analysis. LCM systems consist of
an inverted microscope Wtted with a low-power near-infrared laser. Tissue sections are mounted on standard glass
slides, and a transparent, 100-m thermoplastic Wlm is
placed over the dry section. The laser energy melts Wlm in a
precise location, binding it to the targeted cells, so that individual cells or a cluster of cells can be selected. After the
appropriate cells have been selected, Wlm with adherent
cells is removed, and the non-selected tissue remains in contact with the glass slide. Cells isolated by LCM can be
extracted and submitted to a range of molecular analytical
methods. Thus, mRNA measurements and cDNA microarrays of LCM-puriWed cells from microdissected tissues
allow to compare loss of heterozygosity, detection of mutations and gene expression proWles between various cell
types within a tissue. Mass spectrometric sequencing, peptide mass Wngerprinting, in-gel zymography, and Western
blot have been used to identify proteins of interest. These
approaches are particularly advantageous in identifying the
diVerences between expression levels in normal, developing
and diseased tissues (Curran et al., 2000; Zieziulewicz et al.,
2003).
3. Cytomics in pharmacology and toxicology in vitro
Cytomic analysis may be easily integrated among the
essential strategies to study the interaction xenobiotic-cells
for basic research, industrial development and the evaluation of therapeutic and toxic eVects of chemicals and biological compounds. In general, the aims of applying
cytomics to pharmacology and toxicology can be summarized as follows (Fig. 1):
Fig. 1. Summary of the main processes susceptible of being analyzed by
cytomic techniques in the investigation of the interactions between drugs/
toxicants and cells. The boxes represent major aspects of the interaction.
In italics, particular phenomena that are frequently explored by cytomic
techniques.
3.1. IdentiWcation and selection of speciWc cell subpopulations
Cytomic assays allow to identify and eventually to
purify a particular cell subpopulation of a complex cytome,
by means of Xuorescent staining of surface markers, intracellular functional activities, morphological features or,
more frequently, a combination of the above. As an example of this, by implementing a battery of assays we have
recently demonstrated signiWcant functional diVerences
among hepatoma cell subpopulations previously cloned by
us using a Xow cytometry-based cell sorter on the basis of
diVerential expression of P-glycoprotein in the parental
hepatoma (O’Connor et al., 2005a).
Lately, the multiparametric capacity of cytomic technologies is proven very useful for the identiWcation, characterization and isolation of stem cell populations, including
embryonic, adult and tumoral stem cells. This is of special
relevance, in view of the increasing interest in the cytotoxicity on stem cell populations in toxicological and pharmacological context (Hou et al., 2005). An essential requirement
for FCM in this area is the identiWcation of the so-called
side population (SP), a fraction enriched in totipotent stem
cells in bone marrow (Goodell et al., 1996), human cancers
including leukemia, solid tumors and primary cultures, and
some adult normal tissues (Challen and Little, 2006). In
these models, when cells are labelled with the membranepermeant DNA binding dye Hoechst 33342, a very small
fraction of cells extrudes this dye via ABCG2/BCRP1 transporter and forms a dim tail extending from the normal cell
populations, that allows to purify them via cell sorting.
Technically, SP cells are deWned by the decreased Xuorescence emissions (red, FL7, and blue, FL6) of Hoechst 33342.
The participation of the transporter in the eZux of Hoechst
3342 in SP cells is demonstrated by the disappearance of
G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
179
Fig. 2. An example of the sensitivity of Xow cytometry to detect and to characterize functionally a small subpopulation of relevant cells. (A) Detection of
the side population (SP) of totipotent progenitor stem cells in mouse bone marrow. SP cells are deWned by the decreased Xuorescence emissions (red, FL7,
and blue, FL6) of the vital DNA stain Hoechst 33342. (B) The participation of the ABCG2 transporter in Hoechst 33342 eZux in SP cells is demonstrated
by the disappearance of SP cells following incubation with the eZux blocker verapamil. Plots represent a single experiment using the MoFlo cell sorter
(Dako) and following the procedure described in Goodell et al. (1996).
SP cells following incubation with the eZux blocker verapamil (Fig. 2).
3.2. IdentiWcation of speciWc target cells or cells susceptible
to drugs or xenobiotics
The multiparametric capacity of cytomic methods
allows to identify the expression of receptors speciWc for
given drugs or the presence of structural and functional targets for a drug or xenobiotic within a cell population. In
other cases, relevant parameters are related to the uptake,
retention, biotransformation and eZux of xenobiotic compounds. From such parameters, the sensitivity of a particular cell type can be inferred. Most directly, cytomic assays
can reveal on the whole cytomes or in speciWc subpopulations the eVects produced by the exposure to the drug or
xenobiotic, thus providing evidence for cellular susceptibility or resistance (Lage et al., 2001; Herrera et al., 2003).
Cytomic assays can be also applied to determine the sensitivity of eukaryotic or prokaryotic cells (Fig. 3) that have
been submitted to genomic manipulation, which makes the
cytomic approach an interesting tool to search for new
models in cytotoxicity in vitro (Herrera et al., 2003).
3.3. Detection and quantiWcation of toxicity
Cytomic techniques are widely used for quantitative and
qualitative analysis of cell and organ toxicity (Alvarez-Barrientos et al., 2001). The main advantage of the cytomic
approach derives from their multiparametric capacity
(high-content assays), that provides multiple targets and
endpoints to assess sublethal lesion and death in speciWc
cell subpopulations. Thus, cytomic endpoints may represent early or late marker parameters along the cytotoxic
process. On the other hand, the high velocity of many
cytomic strategies allows the sequential analysis of large
numbers of cells per second, thus evidencing incipient or
minoritary toxic eVects (Alvarez-Barrientos et al., 2001;
Gómez-Lechón et al., 2003).
3.4. Characterization of drug and xenobiotic mechanisms
Because of their multiparametric analytical power, their
temporal (FCM) and topological (CLSM, HCS-B) resolution and the easy interaction with other “-omics”, cytomic
strategies are frequently applied to explore the mechanisms
of actions of drugs and toxics in human, animal and microbial cell models for biomedical (Gómez-Lechón et al.,
2002), biotechnological (Perlman et al., 2004) and environmental studies (Lage et al., 2001).
3.5. Multiplexed analysis of soluble analytes
A recent development of Xow cytometry, multiplexed
assays consist in detecting separately but simultaneously
soluble analytes (usually proteins or nucleic acid sequences)
bound speciWcally by aYnity onto reactive Xuorescent
microspheres of deWned optical properties. The design of
such assays allows to quantify simultaneously several analytes in small volumes of sample. Because of this, multiplexed assays are advantageous over many conventional
biochemical, immunological or molecular bulk standard
techniques (Khan et al., 2004; Fuja et al., 2004).
3.6. High-troughput and high-content screening
The development of current systems of ultrasensitive
detection of Xuorescence, together with the fast rate of data
acquisition provided by interfaced computing systems,
allows the application of cytomic assays to robotized procedures of both high-content and high-troughput screening
of compound libraries, based on cellular Xuorescence in
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G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
Fig. 3. Correlation between genomic modiWcation of bacterial strains (A) and functional cytomic assays by means of end-point (B) or in Xuxo (C)
Xow cytometric determination of intracellular oxidative stress. (A) OxyR, soxR and soxS are essential genes for antioxidant defence in Escherichia
coli, and regulate other downstream genes by sensing hydrogen peroxide (oxyR) or superoxide (sodA, sodB). (B) E. coli B WP4 tester strains that
have been made deWcient in oxyR, sodA and sodB genes exhibit increased levels of intracellular oxidative stress when analyzed by Xow cytometry
using the superoxide-sensitive Xuorochrome hydroethidine, as described in Herrera et al. (2003). (C) OxyR mutants of E. coli B WP4 are more sensitive to hydroxyl radical than wild type controls when exposed to a source of hydroxyl radical (a combination of hydrogen peroxide plus copper sulphate), as shown by in Xuxo assay with the oxidant-sensitive Xuorogenic substrate dihydrorhodamine 123 (DHRh123). The increased rate of
hydroxyl generation in the oxyR mutants over wild type cells is suggested by the enhanced production of Xuorescent rhodamine 123 from the parental leuko dye (DHRh123).
samples deposed in multiwell plates (Perlman et al., 2004;
Smith and Giorgio, 2004).
use succinate or glucose as metabolic fuels (Juan et al.,
1996).
3.7. The in Xuxo level of kinetic analysis by Xow cytometry
4. May cytomics provide in vitro alternative methods for
toxicity assessment?
The dynamic nature of functional cytomic assays is
exempliWed by a unique real-time kinetic approach, the in
Xuxo level (O’Connor et al., 2005b), based on sequential
examination of diVerent single cells from a population
undergoing a dynamic process, that is triggered when
cells of interest are being examined in the Xow cytometer
(Fig. 3). Using the in Xuxo strategy, Xow cytometry has
allowed us to focus on a highly speciWc biochemical process, the activity of the Na+/H+ transporter of plasma
membrane (Dolz et al., 2004). In the other hand, an early
application of the in Xuxo assay provided us a novel
approach to calculate classical parameters of enzyme
kinetics and to use them to reveal similarities and discrepancies between normal rat hepatocytes and rat hepatoma for the mitochondrial bioenergetic processes that
FCM is applied widely to the analysis of in vitro and
ex vivo of many toxicity cellular markers albeit it never has
been applied as a systematic and normalized unique strategy, in the form of an in vitro alternative method for chemical risk assessment. However, the recent developments in
automatization and bioinformatics interfaced to FCM
(Smith and Giorgio, 2004) and HCS-B instruments (Perlman et al., 2004) can be implemented into robotic procedures based on 96-well format assays in equipments
available in growing number of laboratories.
During the development of the European Contract “An
evaluation of the reproducibility and transferability of Xow
cytometric and confocal microscopic endpoints in an
in vitro nephrotoxicity and in vitro metabolism models” we
G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
showed that a compact set of functional assays by Xow
cytometry (the so-called “primary toxicity cytomic panel”,
PTCP) reveals toxic eVects in models of acute, sustained
and delayed exposure of tubular renal cells to nephrotoxins
(cadmium chloride, cyclosporin an cis-platin) in sub-lethal
concentrations (Alvarez-Barrientos et al., 2001). Our results
suggest that cytomic functional assays may detect speciWcally early or transient changes in the process of cytotoxicity, which makes these assays advantageous over other tests
limited to the quantiWcation of cell death as endpoint of
cytotoxicity. In the second part of the mentioned research
contract we showed how the application of PTCP allows to
separate basal- and biotransformation-dependent cytotoxicity by means of the comparison of the cytotoxic eVects of
two neuropharmacological substrates of cytochrome
CYP2D6 (mianserin and imipramine) on the cell line V79
transfected stably with CYP2D6 and its control, mocktransfected cell line (Martínez-Romero et al., 2004). The
Xow cytometric data on nephrotoxicity and metabolismdependent toxicity generated in this study were consistent
with those obtained by confocal microscopy, another powerful cytomic technology, in an independent laboratory
(Alvarez-Barrientos et al., 2001, 2003). Thus, for certain
samples, FCM appears as a versatile tool that allows to
approach diVerent levels of cellular complexity to reveal
cytotoxic eVects and mechanisms in vitro or ex vivo, even in
complex, heterogeneous cell populations.
181
Very recently, our laboratory has been enrolled in the
EC sixth FP Integrated Project “A-Cute-Tox: Optimization
and pre-validation of an in vitro test strategy for predicting
acute human toxicity” (www.acutetox.org). This ambitious
project (37 partners from 13 EC countries) is aimed to
develop a simple and robust assay strategy to predict
human acute systemic toxicity that could eventually replace
current regulatory assays on whole animals. One of the speciWc objectives of A-Cute-Tox is to explore innovative tools
and cell systems to identify new endpoints and strategies
that anticipate better human and animal toxicity. Incorporation of cytomics to this project may deWne new endpoints
of in vitro cytotoxicity to be incorporated into the predictive model or to provide new alerts and correctors of toxicity.
In the Wrst part of this project we have been able of miniaturizing (96-well format) the cultures of rat and human
cell lines, as well as of adjusting the experimental conditions
for toxic treatment, cell resuspension, Xuorescent staining
and Xow cytometric assay with the new Xow cytometers
interfaced to 96-well plate sample loaders. This format of
assay is comparable to that used for most of the current cellular assays of reference for in vitro cytotoxicity (Fig. 4). In
this context, we have deWned the settings and calibration of
the Xow cytometers for this type of assay and established
the correlation in the rat hepatoma Fao between a Xow
cytometric test of cell viability (propidium iodide exclusion)
Fig. 4. Analysis of digoxin cytotoxicity in three established cell lines by a Xow cytometric 96-well automated assay of propidium iodide exclusion. The
established cell lines FAO (rat hepatoma), SH-SY5Y (human neuroblastoma) and A.704 (human kidney adenocarcinoma) were seeded in plastic 96-well
plates and incubated in 10% FCS containing medium for 24 h (50% conXuence) in CO2 incubator at 37 °C. Then, the indicated concentrations of digoxin
in 5% FCS containing medium were added and the plates kept for further 24 h. Then, cells were trypsinized and resuspended in their original wells in 5%
FCS containing medium, in the presence of 5 g/mL propidium iodide (PI) and then analyzed automatically with the Cytomics FC500 MPL multiwellplate Xow cytometer (Beckman–Coulter). Data points represent triplicate wells from two separate experiments. The insert graph shows the Xow cytometric
strategy to diVerentiate live cells (PI excluding cells) from apoptotic and necrotic by means of a plot correlating cell size (forward scatter) and PI uptake.
Notice that neuroblastoma cells are much more sensitive to digoxin than the hepatoma or renal adenocarcinoma cells.
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G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182
and the Neutral Red uptake assay. For all the above considerations, we believe that both the technology (Xow
cytometry) and the strategy (functional cytomic assays)
may succeed in accomplishing meaningfully the proposed
research project.
Acknowledgements
Sponsored by EC sixth FP projects A-Cute-Tox (LSHBCT-2004-512051) and Predictomics (LSHB-CT-2004504761), the Generalitat Valenciana (GV04B-143) and the
Programa de Medicina Regenerativa de la Comunidad Valenciana. E.V. is a Bancaixa post-doctoral fellow. A.G. is
recipient of a Leonardo fellowship.
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