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Cytomics: a Multiparametric, Dynamic Approach to Cell Research 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-inte- grating 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 high- content 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 mol- ecules, 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 tox- icity assessment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Cytometry; Fluorescence; Toxicology; Pharmacology; In vitro 1. Cytomics and cytomes: single-cell based analysis and drug screening, since they approach complex cell-to- in complex systems cell and cell-to-environment interactions, in assay formats that are both information-rich and technically convenient. Genomics, proteomics and metabolomics provide out- In this context, where “-omics” meet Systems Biology, cyto- standing technical contributions to Cell Biology but mics represents a novel analytical strategy aimed to deter- become limited when single or scarce cells are examined or mine multiple biochemical features (the molecular fast cellular processes followed kinetically (Figeys, 2004). In phenotype) in single cells and may be deWned as the cytom- addition, the importance of epigenetic changes (Plass, 2002) etry of complex cellular systems. and the modulatory inXuence of cell environment (Abrous In analogy with other -omics (genomics/genome; proteo- et al., 2005) recommend to combine “-omic” studies with mics/proteome, and so on) the objective of cytomics are the functional analysis (Bernas et al., 2006). Because of this, heterogeneous cellular systems known as the cytomes. The cell-based assays are increasingly sought for basic research cytomes can be understood as the heterogeneous cellular systems and functional components of the pluricellular organisms. Since the functional heterogeneity of the cyto- * Corresponding author. Tel.: +34 963 28 9680; fax: +34 963 28 9671. mes results from both the genome and extracellular envi- E-mail address: [email protected] (J.-E. O’Connor). ronment, cytomics can be considered as a discipline that 0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.07.003 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 177 links genomics and proteomics to cell and tissue function, susceptibility or resistance. The advantages of FCM derive as modulated by external inXuences. Of special importance from its multiparametricity that provides multiple, simulta- is the cell-by-cell basis of cytomic analysis, an approach neous targets to assess cell lesion or death in selected cell that allows to resolve heterogeneous systems and avoids the populations, either as end-point or kinetic measurements. loss of information that characterizes bulk technologies, in which average values are obtained from large number of 2.2. Confocal Xuorescence microscopy cells or from tissue homogenates. For a deeper, comprehensive view of cytomics and their Confocal Xuorescence microscopy is advantageous to relevance to biomedicine, see Valet (2005) and Bernas et al. conventional Xuorescence microscopy, as it narrows the (2006). Weld depth, eliminates out-of-focus blur and allows to pro- duce serial optical stacks from thick specimens (z-axis reso- 2. Technical and analytical features of cytomic technologies lution). Confocality may be applied to image single cells from Wxed or living preparations labelled with appropriate Inherent to cytomics are the use of sensitive, scarcely Xuorescent probes. This technique allows sophisticated cell invasive, Xuorescence-based methods and the integrating information based on spatial- and time-resolved Xuores- concept of individual cell analysis to understand the com- cence measurements and as such is being increasingly used plexity and behaviour of tissues and organisms. Due to the for scientiWc and technological applications (Rubart, 2004; availability of large number of Xuorescent markers and the Ivorra et al., 2006). Usually, CLSM yields better image multiplicity of Xuorescence detectors interfaced to the dedi- quality but the imaging frame rate is slow while SDCM cated instrumentation, cytomic assays may be multipara- may produce video rate imaging, which is required for metric, polychromatic and multiplexed. Fluorescence-based eYcient dynamic observations (Maddox et al., 2003). How- measurements may be qualitative and quantitative and can ever, the new confocal systems function like hybrids allow- be obtained as the result of single end-point measurement ing to obtain both high speed and good resolution. or kinetic, sequential measurements. While these features are common to all cytomic technologies, there are impor- 2.3. Laser scanning cytometry tant speciWc diVerences depending on whether the quantita- tive cell Xuorescence data are extracted together with cell LSC is a microscope-based, scanning cytoXuorimeter morphology in image-based cytomics (Eils and Athale, that combines the advantages of Xow and image cytometry. 2003) or from Xuorescence-pulse analysis in Xow-based It allows multiparametric analysis performed directly by cytomics (O’Connor et al., 2001). Among the current measuring the Xuorescence of individual cells in solid-state cytomic technologies, Xow cytometry (FCM), confocal preparations, such as monolayers, smears, imprints, cyto- laser scanning microscopy (CLSM), spinning-disk confocal spins or tissue sections in several supports including slides, microscopy (SDCM) and laser scanning cytometry (LSC) dishes and multiwell plates. LSC provides increased sensi- are of established relevance. Other cytomic technologies tivity and speciWcity compared to traditional microscopic based on single-cell based image analysis and powerful bio- techniques and a similar structure for data analysis to Xow informatic tools (high-content screening bioimaging, HCS- cytometry, albeit at lower data acquisition velocity. In addi- B) have been recently introduced for drug discovery and tion, it allows relocation of the coordinates of analyzed toxicity testing, as they can provide both high-content and cells of interest following slide restaining. Finally, single high-troughput analysis. Finally, laser capture microdissec- cells identiWed by their Xuorescence measurements can be tion (LCM), a preparative technique for obtaining pure individualized from histogram or dot-plot displays and cells from speciWc microscopic regions of tissue sections, shown as single-cell pictures or combined in picture galler- can also be considered among cytomic technologies. ies 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 An HCS-B system is typically composed by a motorized biological particles) are in suspension. FCM allows the Xuorescence microscope, a CCD camera that captures the simultaneous quantiWcation of multiple Xuorescence emis- images, and a digitizing system that stores the images in a sions in the same cell, arising from Xuorescent markers, and large-capacity computer, with each step controlled by soft- scattered light related to morphology, revealing key cellular ware for image acquisition and analysis. When applied to functions or structures (O’Connor et al., 2001). The velocity high-troughput screening (HTS) cell-based assays this of analysis can be up to thousands of single-cells per second novel technical concept allows cell research at large scale and individual cells from heterogeneous subpopulations using multiwell plates and analyzing in bulk millions of can be physically isolated on the basis of their Xuorescence cells per experiment. These systems may examine up to one or light scatter properties. The multiparametric capacity of single well
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