In the continuing quest to optimize drug discovery, cells and their interaction with media have become the natural focus of assays that can more accurately predict the behavior of the whole organism in response to an agent. Proteomics and contextual biology provide more information about the players in the ligand-receptor and signal cascade relationship, and coupled with the explosion in fluorophore availability and digital processing capabilities, it has become possible to analyze cell dynamics in a far more sophisticated manner.
Spotting Top Performers
High-content screening (HCS) and high-content analysis (HCA) cell-based imaging platforms offer researchers an early look at cell response, protein-protein and cell-cell interactions, and morphological and volumetric changes. Cell-based imaging can help spot top performers in a roster of pharmaceutical leads, as well as provide life science researchers a more integrated approach with which to study the fundamental processes of biology. In addition, as biophotonics applications have matured, it has also become possible to track cellular processes in the whole animal, linking cellome-level data to systems biology, and pushing screening processes to the preclinical level.
Cellomics (www.cellomics.com), a unit of Fisher Biosciences, offers a full HCS/HCA fluorescence-reporting platform that includes instrumentation, software, and reagents.
At the center of its platform are the BioApplications, a collection of HCS algorithms for analysis of various cell-biology measurements, including G-protein coupled receptor (GPCR) signaling, cell-health profiling (apoptosis/necrosis; early and late toxicity; survival of target-expressing cells), molecular translocation, analysis of cell morphology (including inter- and multi-cellular changes), neuronal profiling, and automatic identification and quantification of micronuclei.
HitKit® HCS Reagent Kits are available for all BioApplications, allowing researchers to quickly develop assays with optimal reproducibility, according to Cellomics. Each kit contains the requisite fluorescent reagents, buffers, and optimized sample-preparation protocols. The imaging systems integral to these products are the ArrayScan® and KineticScan®, HCS Readers, and the cellWoRx™ High Content Cell Analysis System. These systems are optimized for both fixed-endpoint and live-cell kinetic studies.
Judy Masucci, Ph.D., director of marketing and sales support at Cellomics, describes the ArrayScan VTI HCS Reader as the company’s flagship HCS system, designed for the screening environment. Both ArrayScan and KineticScan “offer on-the-fly image analysis, addressing the imaging bottleneck—acquisition of images. It saves time in most cases by allowing the user to add intelligence to the image process. For instance, you can instruct the software to keep imaging until you get a hit, or count the number of hits per well. You can also implement stop criteria (useful if several wells are bad), or program rare-event assays.”
In addition, the user can build statistical analysis into the screening process, which saves post-processing time. The High Content Informatics (HCi™) suite of software “handles volumes of data, which can encompass hundreds of parameters per cell, per well, and per field.”
Dr. Masucci explains that Cellomics “has made arrangements with competitive companies to use Cellomics’ HCi platform through an interface known as HCS Gateway™. The HCi suite is comprised of a database called Cellomics® Store (for both images and data), an HCS Applications server (middleware), and the Gateway itself (instruments that talk to the middleware).”
The vHCS™ Discovery Toolbox, which is also a part of the HCi suite of software, enables users to visualize and make decisions from their data. A version of this toolbox, known as the vHCS Analysis Toolbox, can also be used in tandem with the cellWoRx system, a fully integrated HCS imaging system that is based on Applied Precision’s (www.appliedprecision.com) illumination pathway and offers four-color, rapid image acquisition, ranging from UV to IR.
For ease of use Cellomics offers turnkey solutions such as prescripted parameter settings and tailored algorithms, benefiting either the inexperienced or well-versed experimentalist.
BD Biosciences (www.bdbiosciences.com) offers an array of imaging systems, ranging from a basic microscopy package to a system designed to image live cell kinetics and other time-resolved events, looking at the cell in the context of its matrix or substrate. In the middle range of capability is the BD Pathway™ 435 series benchtop imager that offers eight excitation, eight emission, and five dichroic filter positions that can be independently controlled to produce a broad array of fluorescence detection.
Its fluorescence-based bioapplications include, among others, neurite outgrowth, protein translocation, angiogenesis, colocalization, cell cycle and 3-D confocal analysis. Phil Vanek, Ph.D., director of global marketing, explains that BD Biosciences’ approach to “bioimaging complements our flow-analysis capabilities. While cytometry is a sensitive and flexible powerhouse technology, bioimaging is helpful when cells are investigated in a contextual format—grown on novel substrates or in clusters.
“The BD Pathway 855 is a high-content platform geared to automating high-resolution functional cell-based assays,” enabling such experimentation as “fast calcium kinetic measurements, time-lapse experimentation, 3-D exploration of cell clusters, ADME and toxicity applications, and screening of antibodies.”
The company also offers the BD FACSArray™ Bioanalyzer, a fully integrated two-laser flow analyzer that can perform cellular or cytometric-bead based analysis. With over 1,500 available reagents and the ability to capture up to 15,000 events per second, the Bioanalyzer covers the full range of biological applications, including intracellular phosphorylated protein detection and cytokine analysis and cell-cycle events. The unit features a microtiter plate sampler, and is designed for use with BD’s Cytometric Bead Array (CBA) Flex Set. Dr. Vanek adds that the “customizable bead array technology enables researchers to develop their own uniquely configured experiments.”
The Animal Model
Xenogen (www.xenogen.com) offers VivoVision®, based on luciferase illumination in cells, microorganisms, and animals. This model allows direct visualization of target validation, efficacy, pharmacokinetics, lead optimization, ADME, and safety/toxicology parameters from the molecular to cellular to whole animal level. According to Xenogen, its technology allows real-time analysis of in vivo metabolism, offering numerous advantages in the scale and complexity of information and analysis, and potentially reducing the time to market for a compound.
Pamela R. Contag, Ph.D., co-founder of Xenogen, says that she “strongly believes that the cell-based assay should be linked to the whole animal. In vivo imaging will help not just our biotechnology customers, but also academic and life science researchers develop models so that people can look at the physiology of the whole animal, whether you are looking at target interactions with drugs or at the disease state alone.”
In the animal model, “the researcher can look at upstream and downstream events in the cascade, whether by labeling a specific gene, building a protein chimera, or by examining specific protein expression.”
As one of the four available IVIS® imaging systems, the Xenogen IVIS® 3-D and supporting software constitute a biological systems-based approach that will enable researchers to “employ a discipline for robustness to get clues about specific types of toxicity that occur with novel biologics,” concludes Dr. Contag.
Xenogen was recently acquired by Caliper Life Sciences (www.caliperls.com). According to Mark Roskey, Ph.D., vp, reagents and applied biology, “with Caliper’s technology you can pinpoint a signal from a single cell, testing up to 12 samples at a time on what is essentially a flow cytometer on a microchip. For higher content screening, Caliper is working to develop a cell assay plate, combining microfluidics with a microplate format. This assay will allow generation of real-time dose-response curves with 12 different doses, and is expected to be launched in early 2007.”
Caliper is already working with key customers on both its micro-fluidics platform and the Xenogen IVIS imaging systems, and Dr. Roskey points out an early success in therapeutics with this combination of technologies. With Gleevec and other kinase inhibitors (KI) coming under recent scrutiny for possible cardiac effects, Caliper and Xenogen “combined technologies to provide a set of in vitro to in vivo platforms for testing a new KI for possible side effects and in vivo efficacy. Pfizer’s Sutent, for example, was recently approved by the FDA for the treatment of stomach cancer—and some of the early dosage studies were accelerated by quantitating tumor growth inhibition in vivo using the Xenogen imaging platform.”
In addition, Dr. Roskey notes that “because it is becoming more critical to profile KIs against others for selectivity, Caliper has been working to develop rapid selectivity profiling, specifically for kinases. A new product, ProfilerPro, will provide 48 kinase assays using microfluidic technology,” to provide a clear picture of kinase selectivity early in the screening process.
Molecular Devices (www.moleculardevices.com) offers a fully integrated HCS screening platform based on two inverted microscopes, ImageXpress® Micro and ImageXpress Ultra. Jan Hughes, vp of worldwide marketing, says that Molecular Devices also provides a “flexible image-acquisition software system to address the wide variety of existing assays, as well as less typical applications such as measuring the heart rate of zebrafish. The most difficult component is image analysis: in a given day you may develop 30,000 images, and each image may have 100 cells with over 30 data points per cell.”
For tying these large data volumes into a database, “AcuityXpress is a cellular informatics data-management tool that simplifies the process of extracting data, choosing controls, and matching data points to controls. In addition, MetaXpress acquisition software includes application modules that offer different options to suit different types of research. An expert could buy the software and customize it, or a non-expert could just buy a neurite outgrowth module,” says Hughes.
“In addition Molecular Devices offers MDCStore, a free, fully scalable, enterprise-level high-content database that any customer can integrate with their corporate database structure, making possible a seamless and integrated flow of data from assay design and compound libraries to image analysis and data-mining results.”
Beckman Coulter (www.beckmancoulter.com) Cell Lab Quanta™ SC is a “true flow cytometer that can run pretty much anything from a particle to a cell, or bead-based chemistry,” explains business manager Brendan Yee. The cytometer is designed to deliver contextual biology data and also employs the Coulter volume, which “accurately measures cell size by electrical impedance, essentially by displacing a certain volume of electrolyte.” Yee adds that when analyzing cell response to an agent, “many of the toxicity mechanisms will cause a cell to shrink, and a 10% change in cell diameter can represent a two-fold change in cell volume, a very sensitive indicator. Other flow cytometers do not give a true accurate reading and must run standards to get a concentration of cells/mL.”
The cytometer can screen for “cell expression, protein-protein interaction, knockdown of a particular fluorescent protein or effects of siRNA, and examine such processes as viability, cell cycle, apoptosis, and CD markers.”
The company is currently in the process of launching a multiplate loader for the Cell Lab Quanta SC, adding the ability to handle flat, round, or deep wells, 4-mL cups, and Eppendorf tubes.