Screening
New tools increase accessibility to HCS ASSAYS.
New Tools Increase Accessibility to High Content Screening Assays
By Dr John Comley
Summer 2012

There have been major improvements in the instrument offerings and tools that enable high content screening (HCS) over the past decade.

In this review we examine the status of high content screening assays today; the motivation to purchase assays kits versus developing homebrew; the use of specialty microplates; spending on high content screening assays, reagents and consumables; and what is needed to expand the market for high content screening assays.

This is explored with respect to enhanced vendor offerings in HCS-engineered microplates, microchannel plates, new cell models incorporating high content screening, new assay dyes and reagents, and new high content screening assay kits. We conclude that HCS assay development has never been more accessible and supported by such a diversity of off-the-shelf products.

High content screening (HCS) has progressed a lot in the past decade, with countless improvements in the instrument offerings and supporting tools. Although advances in software, automation, microplates and imaging hardware have now permitted high content imaging to expand into higher throughput applications, it is still not comparable to other HTS technologies, in terms of numbers of wells processed (1). This reflects both the cost and complexity of performing high content screening assays, particularly if live cells are imaged.

Vendors now offer tool box reagents, for example, validated antibodies to monitor signalling from just about any pathway in a cell with the specific mutations and cellular signalling that best matches the disease targeted. Antibodies can also be screened in panels for multiplexed staining to answer key questions more quickly, such as whether a compound is getting into a cell and whether it acts on its intended target. Vendors are also moving towards greater off-the-shelf offerings in terms of high content screening assay kits, increasingly supplied with specific cell lines, pre-configured analysis routines and available as components.

Although many consider homebrew, ie developing and validating your own high content screening assays, is the best way to get the results you need, end-users are starting to understand that these are not trivial projects. Building up the required capacity and expertise in optimising the necessary antibodies and assay validation is time-consuming and expensive, particularly for the academic community. As was the trend with other emerging screening technologies, labs now investigate what is available offthe- shelf first, before developing assays from scratch.

In this review, based on HTStec’s high content screening Assays, Reagents & Consumables Trends 2011 market report (2), we examine the status of high content screening assays today; the motivation to purchase assays kits versus developing homebrew; the use of specialty microplates; spending on high content screening assays, reagents and consumables; and what is needed to expand the market for high content screening assays. These findings are reported together with the latest vendor offerings in high content screening assays, reagents and consumables.

Current status of high content screening

Survey respondents reported a median of four to five different high content screening assays run per year, each with a median of 5,000 to 10,000 wells analysed per high content screening assay. Other recent surveys exclusively of high throughput screening labs (1) have suggested that the total number of high content screening primary screening wells run per year is about 10% that of other cell-based assays, although the use of high content screening in primary screening is growing faster than most other assay readouts.

In addition, there is some suggestion that hits identified through high content screens translate more efficiently into leads and nominated candidates compared to hits identified through non-HCS cell-based assays and biochemical screens.

The main use of high content screening by survey respondents was drug discovery (75% using). This was followed by mechanistic studies (56% using) and studying cell behaviour or differentiation (55% using). Least used today was safety/toxicology (37% using) (Figure 1).

Figure 1 Main uses of high content screening assays

Interest in broad high content screening categories was ranked. This analysis showed greatest interest for cell viability/ toxicity.This was very closely followed by cell signalling and then receptor binding/translocation. Least interest was shown for stem cell differentiation (Figure 2).

Figure 2 Interest in broad high content screening assay categories

The cell type most used today (2011) for high content screening assays was transformed or recombinant cell lines (48% of assays). This was followed by primary cells (20% of assays); transiently transfected cells (13% of assays); stem cell-derived phenotypes (6% of assays); diseased primary cells (6% of assays); isogenic cells (4% of assays); and undifferentiated stem cells (3% of assays) (Figure 3).

Figure 3 Percentage of high content screening assays done with different cell types

Accessing high content screening assay reagents

The majority (67%) of high content screening assays developed by respondents were homebrew versus reagents purchased in assay kit form. Validated protocol was rated the most important reason to purchase reagents in assay kit form. This was followed by saves time – gets an assay up and running quickly, and then the technology advantage of a kit (eg specificity, sensitivity or speed). Rated least important was bulk purchase (Figure 4).

Figure 4 Main drivers to purchase high content screening reagents in assay kit form

The high content screening assay categories most likely to be purchased as reagents in assay kit form were cell viability/ toxicity (69% might purchase). This was followed by cell signalling (54% might purchase) and then receptor binding/translocation (39% might purchase). Least likely to be purchased was cell migration (28% might purchase) (Figure 5).

Figure 5 high content screening assay categories most likely to be purchased as reagents in assay kit form

The majority (73%) of respondents were interested in getting preconfigured analysis routines with the purchase of high content screening assay kits. Respondents rated assay not available in kit form as their main driver to purchase high content screening reagents separately and to develop homebrew assays. This was closely followed by specificity of the assay and then ability to combine with more parameters/customise. Equally rated least important drivers were availability of protocols from peer reviewed publications and limited assay numbers (Figure 6).

Figure 6 Main drivers to purchase high content screening reagents seperately and to develop assays homebrew

Microplates used in high content screening

The density of microplates most used for high content screening applications by respondents were 96 and 384- wells. Most of these microplates had plate bottoms made of plastic film. Most plates were, however, HCS-engineered (ie had features that enhanced their performance in high content screening) and had a tissue cultured-treated surface. The majority (84%) of respondents were, however, not using 3D cell culture scaffolds or microfluidic components in their microplates prior to or directly in high content screening assays.

Survey respondents most positively agreed with the statement ‘Glass-bottom microplates are too expensive in comparison to optical-grade plastic-bottom microplates’; moderately agreed with the statement ‘HCS engineered microplates yield higher quality data than regular microplates’; and slightly disagreed with the statement ‘Glass-bottom microplates are more suitable to high content screening than optical-grade plastic-bottom microplates’ (Figure 7).

Figure 7 Opinion on the following statements about high content screening microplates

Spending on high content screening assays, reagents and consumables

Survey respondents had a median budget allocated of $50,000-$100,000 per lab for spending on high content screening assays, reagents and consumables in 2011. The biggest proportion (36%) of this budget was allocated to accessory reagents (toolbox components, stains, Abs, expression tags, fixatives, buffers etc). This was followed by 20% microplates; 19% HCS assays kits; 8% image analysis software; 6% HCS cell lines; 6% high content informatics; 3% outsourced assay or cell-line development and 2% other spending (Figure 8).

Figure 8 Breakdown of spending on high content screening assays, reagents and consumables

What is needed to expand the market for high content screening assays

Survey respondents ranked the aspect of high content screening most limiting (preventing the use of more applications) as physiological relevance of assays. This was closely followed by throughput too low or reagent/assay cost (both equally ranked) and then software/analysis is limiting (Figure 9).

Figure 9 Aspects of high content screening that are most limiting

Physiologically relevant cell culture methods were the area of development rated most needed to grow the market (increase use and adoption) for high content screening assays. This was followed by software/algorithms, lower costs and then 3D cell cultures. Least needed was better microplates (Figure 10).

Figure 10 Areas of development needed to grow the market for high content screening assays

Latest developments in tools for high content screening

The following vendor snapshots provide additional details and describe some of the latest developments in microplates, assay kits, reagents and other tools used for high content screening:

The Vision Plate™ from 4titude® (www.4ti.co.uk) has been designed for high content screening assays in drug discovery and related areas. It is also suitable for homogeneous assays employing fluorescence intensity, FRET and TR-FRET where measurements are bottom- read. This high quality optical base plate assures the necessary accuracy and consistency for automated high throughput systems, generating optimum signal-to-noise ratios.

The use of laser welding to assemble the plates reduces base film distortion during production, therefore improving base flatness. Additionally, laser welding reduces auto-fluorescence, when compared to other techniques used to assemble plates, such as glueing. The range consists of 384-well, 96-well and 24-well plates. They are available with either a 150μm or a 700μm polystyrene base. For top-read fluorescent or luminescent assays, 4titude also offer the plates with a solid bottom in black or white (Figure 11).

Figure 11 The 4titude 96-well Vision PLate viewed from the top

The extensive BRAND Life Science range (www.brand.de) includes products for PCR, storage, microplates for immunology and cell culture as well as pipette tips, filter tips, microcentrifuge tubes and UV-transparent disposable cuvettes. The wide range of 96-, 384- and 1536-well microplates from BRAND (known as BRANDplates®) are available in different bottom shapes and materials. Our surface treatment technology results in eight different surface types to fulfill the needs of a broad range of applications. These include a nontreated surface, three new immunological and four new cell culture surfaces.

The new product line covers a multitude of standard applications (eg homogenous screening assays) as well as applications in the fields of immunology, cell culture techniques and high content screening. New in the range are the 96-well strip plates, designed to fit any application. Strip plates are available without or with a grid to facilitate the use of small numbers of wells in versions with high-binding or medium-binding capacities (Figure 12).

Figure 12 BRANDplates are now offered in eight different surface treatments, four for cell-based assays

The Aurora™ range of cyclo-olefin polymer (COP) plates includes clear bottom plates ideal for high content imaging applications.

Figure 13 demonstrates many of the benefits of these plates. The image was taken using the Celigo® cell imaging system from Brooks Life Science Systems (www.brooks.com) and shows the clarity and consistency of the well bottom on this Aurora black 1536-well plate with square well, low base, 188μm film, tissue culture treatment and sterility.

Figure 13 A black 1536 square well of an Aurora COP plate from Brooks Life Science Systems

This has always been a strength of the Aurora range; however, now that both Aurora and Celigo are Brooks Life Science Systems products, the Celigo is being used in quality and continuous improvement programmes to further improve quality on Aurora plates. The latest imaging instruments, such as the Celigo, have the advantage of ‘brightfield’ imaging, with consistent illumination right to the edge of the well; this imposes new demands on microplates. Cells typically tend to grow at the well wall. This demands a distinct edge between the well wall and the well base.

The moulding technique used for Aurora plates means that the plate frame is welded, during the moulding process, to a high quality COP film base, not only giving high optical clarity and a consistent image, but also a clean base/well wall boundary. Other techniques, such as glueing the base to the frame, frequently leave residue that makes it difficult to identify cells at the edge of a well.

GE Healthcare’s (www.gelifesciences.com/cardiomyocytes) human embryonic stem cell (hESC)- derived Cytiva™ cardiomyocytes are a cell-based high content model that provides a physiologically relevant and sensitive alternative to current cell models for predictive toxicity testing. Cytiva cardiomyocytes are derived from hESCs with a normal karyotype and represent a typical adult human myocyte population (around 80% ventricular myocytes and including atrial and nodal subtypes).

Once thawed and cultured, the cells form a contractile monolayer and express cardiac-related transcription factors and cardiac structural proteins. A good cardiac cell model must reflect the electrophysiology of the heart, and the Cytiva cardiomyocytes express all the major sodium, calcium and potassium ion channels, including the hERG potassium channel. The channels in the cardiomyocytes are at the levels normally seen in human heart tissue and respond reversibly to ion channel blockers.

In predictive toxicity testing, high-content imaging and analysis can be used to find nonarrhythmic effects that could disrupt normal cardiomyocyte function. Signs of toxicity include changes in cardiomyocyte morphology, nuclear structure and content, plasma membrane and mitochondrial structural integrity, cell-cell junctures and intercellular communication, calcium mobilisation and mitochondrial respiration.

Because Cytiva cardiomyocytes closely mimic authentic human cardiomyocytes, drugs can be tested at clinically relevant concentrations, and thus have the potential to predict the toxicity in humans more accurately than other cell types. GE Healthcare has also developed a reliable and reproducible process that produces large quantities of Cytiva cardiomyocytes in an efficient and costeffective manner (Figure 14).

Figure 14 hES cell-derived Cytiva cardiomyocytes from GE Healthcare in a live multiplexed toxicity assay

The advent of high content screening as a technology for microscopic analysis of cells has changed the requirements for consumables in drug discovery. Microscopic systems have been optimised to improve sensitivity, accuracy and ease of use. These specific needs had a major impact on state-of-the-art platforms requesting microplates with an excellent chemical resistance and outperforming optical and physical properties. A new set of polymers, namely COC and COP fulfill these requirements.

Greiner Bio- One’s (www.gbo.com) recently launched 1536-well SCREENSTAR microplate is a cycloolefin microplate designed for microscopy, high-content screening, and high-throughput screening. This microplate features a black pigmented frame with a 190μm ultra-clear film bottom as a perfect match for compatibility with instrument optics.

Well bottoms display excellent optical properties for the highest optical transparency, with reduced autofluorescence, low birefringence and a refractive index similar to glass. Recessed wells enable complete periphery access for high-magnification lenses. Cell culture treatment and sterility assure exceptional performance for high-content screening. A smooth microplate top, absent of alphanumeric coding, facilitates flush lid mounting for use with ultrahigh- throughput screening systems.

As HCS will be more widely used in the future, the range of plates will be expanded to very similar plates with 384 wells and a plate in a 96-well format with round wells minimising shadows and the meniscus effect. In addition, this plate will have a ditch at the perimeter of the plate and can be filled with media or water to reduce any kind of edge effect (evaporation) at prolonged incubation times (Figure 15).

Figure 15 Greiner Bio-One's 1536 well SCREENSTAR plate for high content screening

Matrical Bioscience (www.matrical.com) is a leading supplier in the life-science research market through the development of innovative, automated products targeted for drug discovery and genomics applications, offering consumable microwell plates in 96, 384, and 1536 formats (MatriPlates). MatriPlates combine the benefits of glass-bottomed plates, low background fluorescence, and no crosstalk, to provide the highest possible optical Drug Discovery World Summer 2012 High Content Screening quality for imaging cell-based assays.

Available in a choice of highly uniform 0.17mm or 0.72mm plate bottom thicknesses, the glass surface within each well is scratch-resistant and prevents the formation of microbubbles providing a flat and optically clear surface that is ideal for the study of cell growth, providing the highest level of error-free performance with all high-content screening assay instrumentation (Figure 16).

Figure 16 Matrical Biosciences' glass-bottomed 96-well MatriPlate microplates enable high quality imaging for high content screening

The Transfluor® Cell-Based GPCR Assay from Molecular Devices (www.moleculardevices.com) is designed to screen G-protein-coupled receptors (GPCRs), ligands and other potential drugs that regulate GPCRs. It is compatible with commercially available high content screening systems, allowing for rapid detection of compound activity against known GPCR targets. The patented technology traces fluorescently-labelled-arrestin that binds to GPCR upon receptor activation and marks it for internalisation via clathrin-coated pits. Hence monitoring the redistribution of -arrestin can provide insight into GPCR activation, translocation, degradation and recycling.

The Transfluor technology has been validated as a gold-standard high content screening assay for drug discovery. A winning feature of the Transfluor Assay is that it requires no prior knowledge of the interacting G-protein, which makes it suitable for orphan GPCR screening. The Transfluor technology is validated with more than 100 GPCRs and across all GPCR classes regardless of the interacting G-protein. A single read-out is compatible with all GPCR subtypes, thus eliminating the need for multiple GPCR assays.

Depending on user’s needs, time-limited, trial period evaluation kit and tiered licensing options are available to cover needs from basic research up to highthroughput screening. The Transfluor Assay has been optimised to run on ImageXpress® Micro Widefield and ImageXpress Ultra Confocal high content screening systems. Powered by the MetaXpress® Transfluor Software Application Module, pits and vesicles with -arrestin can be imaged and quantified, providing a basis for universal, high-throughput, high-content analysis for all GPCRs (Figure 17).

Figure 17 GPCR activation using the Transfluor Assay and imaged with ImageXpress Micro System from Molecular Devices

The toolbox of reagents for high-content analysis from Molecular Probes® (www.lifetechnologies.com) regularly expands by additions of assay kits and reagents. Novel kits for interrogation of cell health and cytotoxicity include green versions of the fluorogenic pHrodo™ pH sensor, CellROX® oxidative stress sensor and CellEvent™ caspase 3/7 assay kit. They spectrally and functionally complement its existing offerings for HCS platforms, including assays for apoptosis, autophagy, cell cycle, cytotoxicity and intracellular trafficking.

CellROX®, pHrodo™ and Cell- Event™ reagents are compatible with several instrument platforms, but Molecular Probes also offers assays and reagents tailored specifically for high content screening platforms. For image analysis strategies based on nuclear segmentation, the HCS NuclearMask™ stains are available in three different colours and stain live or fixed cells; the HCS NuclearMask™ Red Stain is ‘tunable’ for nuclear versus cytosolic intensity by optimising the concentration. For image analysis strategies requiring whole-cell segmentation, the HCS CellMask™ stains label the entire cell (ie cytoplasm and nucleus) and are applied to cells immediately after fixation and permeabilisation.

Available in five different colours, the HCS CellMask™ stains are bright and photostable with narrow emission spectra. The FLoid™ Cell Imaging Station was recently added to Life Technologies’ line of benchtop instruments. It is a fully integrated device with an intuitive interface that seamlessly combines high-quality optics, detection in three fluorescence channels (DAPI, FITC and Texas Red filters) as well as transmitted light.

It features a fixed, high-quality 20x plan fluorite objective, with additional magnification obtainable using the digital zoom. The streamlined image acquisition process and real-time multicolour display allows high-quality images to be captured with a few mouse clicks, making it an ideal tool to quickly check fluorescence labelling quality and/or cell confluence (Figure 18).

Figure 18 Morphological changes and cytoskeletal rearrangements in HeLa cells after cytochalasin D treatment

As researchers are striving to use cellular models which more closely resemble the situation in vivo, detection of targets expressed at low levels is becoming increasingly important. PerkinElmer (www.perkinelmer.com) has therefore launched the HCA ImageAmp™ reagent kits. Based on PerkinElmer’s proprietary Tyramide Signal Amplification™ (TSA) technology they deliver unprecedented sensitivity for antibody-based high content screening assays without compromising resolution.

TSA amplifies target signals by covalent deposition of numerous fluorophores proximal to the target of interest. Depending on the requirements of the assay HCA ImageAmp™ kits can be used to significantly improve detection levels, eg of phospho-Histone 2AX or to reduce consumption of primary antibody. The quest for more biologically relevant model systems leads to an increasing interest in complex cell culture systems and in vivo validation. PerkinElmer has developed a range of targeted and activatable fluorescent agents for in vivo imaging, some of which can be applied to phenotypic high content screening.

One example is looking for cathepsin activity in cancer cells. As cathepsins are upregulated during metastasis progression, they are useful biomarkers for cancer research. Upon activation by cathepsins the ProSense™ imaging agent from PerkinElmer is emitting a fluorescent signal which can be quantified in cultured cells and in vivo. The ability to utilise the same agent under both conditions improves the comparability of results acquired in increasingly complex model systems and offers a means for direct translational research (Figure 19).

Figure 19 Activation in viable HT-29 human colorectal adenocarcinoma cells with PerkinElmer's ProSense 680 imaging agent

The Thermo Scientific (www.thermoscientific.com) portfolio of reagent products for the high content market leads the way in providing the most convenient and easy to use kits and consumables for all platform providers in the high content space. Compatible with most imaging platforms, the Thermo Scientific Cellomics Reagent Kits and newly-introduced kit components meet the demand for validated out-of-the-box tools while retaining the ability to order in bulk or custom volumes for those using high content platforms and cell-based screening laboratories.

As better tools are needed for automating chemotaxis and migration assays, Thermo Scientific now offers the BellBrook iuvo™ Microchannel 5250 Migration Assay Plate and the iuvo™ Chemotaxis Assay Plates. The advanced technology in the iuvo™ Microconduit Arrays enable the use of highly miniaturised, advanced cell models and functional assays with automated liquid handling and HCA platforms, with the goal of more accurate replication of in vivo processes in drug discovery.

Instead of the conventional ‘buckets’ of various sizes used for cell culture in multiwell plates, iuvo™ plates have cell culture compartments with geometries designed specifically to support the biology and/or functions of interest. Thermo Scientific also offers the Nunc Edge Plates which are ideal for screening applications where it is desirable to reduce assay edge effects.

The Thermo Scientific Cellomics Redistribution technology provides stably transfected cell lines for more than 80 targets, perfect for high content screening campaigns. When Thermo Scientific’s portfolio of consumables is combined with its leading high content imaging and software tools, it enables researchers to expand the depth and breadth of their cellular research (Figure 20).

Figure 20 The BellBrook iuvo plates from Thermo Scientific

Summary - New Tools Increase Accessibility to High Content Screening Assays

From the vendor snapshots it is possible to highlight several key areas where high content screening tools have recently undergone significant enhancement:

High content screening engineered microplates: Many features have now been incorporated into microplates to provide the highest level of error-free performance with high content screening instrument platforms.

These include:

1) glassbottoms to provide the best possible optical clarity together with a flat surface that prevents the formation of microbubbles

2) high quality optical polystyrene film bases giving reduced autofluorescence, low birefringence and a refractive index similar to glass

3) laser welding to reduce auto-fluorescence associated with glueing and to create a clean base/well wall boundary

4) new COC and COP polymers giving enhanced optical clarity and excellent chemical resistance

5) recessed wells to enable complete periphery access for high-magnification lenses

6) a smooth microplate top to facilitate flush lid mounting

7) a perimeter ditch that can be filled with media or water to reduce edge effects (4titude, Brand, Brooks, Greiner, Matrical, Thermo)

Microchannel plates: Microconduit array plates enable the use of highly miniaturised, advanced cell models and functional assays with automated liquid handling and imaging platforms. The iuvo™ plates have cell culture compartments with geometries designed specifically to support automated high content chemotaxis and migration assays (Thermo).

New cell models incorporating high content screening: Physiologically relevant and sensitive alternatives to current cell models for predictive toxicity testing that utilise multi-parameter high content screening, eg hESC-derived cardiomyocytes providing a cardiotoxicity model (GE Healthcare). New assay dyes and reagents: Some of the new offerings include: HCS NuclearMask™ stains available in different colours to stain live or fixed cells and ‘tunable’ for nuclear versus cytosolic intensity by optimising the dye concentration (Molecular Probes).

HCA ImageAmp™ reagent kits to enhance the detection sensitivity for antibody- based high content screening assays (PerkinElmer). A range of targeted and activatable fluorescent agents for in vivo imaging, some of which can be applied to phenotypic high content screening, eg the activation of the ProSense™ imaging agent by cathepsin activity in cancer cells (PerkinElmer). New high content screening assay kits: Cellomics Reagent Kits and newly-introduced kit components are validated out-of-the-box tools to meet the needs of all high content labs whatever their requirement (Thermo).

The well-established gold-standard Transfluor and Redistribution high content screening assay technologies are now more accessible through wider offerings and flexible licensing options (Thermo, Molecular Devices). More alternatives are available as novel high content screening kits for interrogation of cell health and cytotoxicity based on pHrodo™ pH sensor, CellROX® oxidative stress sensor and CellEvent™ caspase 3/7 assays are launched (Molecular Probes).

In conclusion, based on the current availability of an assortment of highly specialised tools, high content screening assays have never been simpler to develop and easier to screen. DDW

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Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation and assay reagent technologies) to drug discovery and the life sciences. Since its formation nine years ago, HTStec has published more than 80 market reports on enabling technologies and Dr Comley has authored more than 35 review articles in Drug Discovery World. Please contact info@htstec.com for more information about HTStec reports.

References
1 Future Directions of HTS Trends 2012 Report, published by HTStec Limited, Cambridge, UK, April 2012.

2 HCS Assay, Reagents & Consumables Trends 2011 Report, published by HTStec Limited, Cambridge, UK, October 2011.

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