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Thinking small: MINIATURIZATION MEANS MORE THAN LOW VOLUMES
12-28-2005
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By Wayne P. Bowen, Sarah L. Payne and Ben J. Schenker
 
Assay miniaturization in high-throughput screening (HTS) has been adopted by the pharmaceutical industry to accelerate the R&D cycle and to reduce costs. To improve further, there is now a drive to increase the quality of hits from the primary screening stage by providing more information, earlier. To do this effectively, cell-based assays producing high-content information for target identification and validation must be miniaturized so they are suitable for an HTS environment. However, this raises issues with assay viability and robustness which need to be addressed and requires specialist technology for liquid handling, plate preparation and analytical instrumentation. This article provides an overview of some of the innovative technologies available to enable high-content screening (HCS).
 
The main drivers for assay miniaturization are increased throughput, greater efficiency and reduced reagent/consumable usage. Contemporary compound inventories regularly hold over a million compounds and, with an increasing number of therapeutic targets emerging, miniaturized formats are essential to cope with so many permutations. A 16-fold increase in throughput is achievable by moving from 96- to 1536-well formats, providing assay configuration and analysis are not rate limiting. Reducing assay volumes to a few microliters decreases the amount of assay constituents required, thus extending the lifetime of valuable chemical and protein libraries and also reducing the demand for expensive materials, particularly cell cultures. Cost savings achieved from assay miniaturization may also permit a broader profile of pharmacological activity to be tested within HTS screens, generating invaluable data on compound specificity.
 
By incorporating the benefits of miniaturisation into cell-based assays, high-content information can begin to be obtained during primary screening. This enables more compounds to be eliminated, to give smaller, more specific, sample sets for hit-confirmation and secondary screening. This ensures a rapid decision-making process resulting in more efficient drug discovery.
 
 
High-content analysis
 
Assay miniaturization does not simply involve the use of high-density plate configurations and decreased assay volumes. These factors have a knock-on effect on the dynamics of assay reactions, set very significant challenges for accurate plate preparation and require the use of sensitive readers.
 
The physico-chemical properties of smaller wells bring a new set of problems, especially for cell-based screening, as cells are intrinsically very sensitive to environmental changes. For example, evaporation may need to be tackled by considering humidity levels, plate lidding or sealing. In addition, using lower cell numbers can lead to a decreased assay signal or to less reproducible data due to heterogeneity of cell response. Most CCD image-based HCS systems sample too few cells to generate robust assay results, and are too slow for HTS, especially when using high-density plates. Another solution for maintaining assay sensitivity and robustness is to use a laser-scanning microplate cytometer to analyze individual cell responses for all cells and report the percentage of responders in each well. Instruments such as the Acumen Explorer (TTP LabTech) can scan and analyze an entire 1536-well plate in 10 minutes, thus maintaining the throughputs required while providing high-content information.
 
 
Liquid handling
 
Assay miniaturization places a large burden on liquid handling systems. Few instruments combine speed with accurate nano- to microliter pipetting capability, whilst ensuring zero cross-contamination. Addressing high-density plate formats also presents problems for many. For true flexibility, instruments should also cope with solutions of differing viscosities and leave negligible dead volumes to maximise the cost benefits of applying low assay volumes. For HCS, liquid handlers must be capable of pipetting cells in low volumes while maintaining their viability. This can encompass adding as few as 50 cells per well in high-density plates for assays involving prolonged culture over several days.
 
The preparation of assay-ready plates is becoming key to maximizing throughput for many HTS departments. These plates contain test compounds prepared from library stocks ahead of time at the correct concentrations. Assay-ready plates save time and consumables by eliminating the need for intermediary plate preparation between compound store and screening laboratory. DMSO is routinely used for compound dissolution, but its presence at concentrations above 1% can markedly affect assay performance. Thus, accurate nanoliter quantities of DMSO-based stock solution are required to keep concentrations low when preparing assay-ready plates. This direct use of stock solution eradicates uncertainty about compound stability when prediluted in aqueous solutions, but means ensuring zero cross-contamination is critical. Cross-contamination has been shown to result in the appearance of artificially efficacious compounds, or false positives/negatives. Liquid handlers using disposable tips eliminate cross-contamination but have an associated consumable cost. Non-disposable pipetting heads can offer high throughputs, but require very stringent wash steps to avoid problems when transferring some compounds.
 
The different liquid handling technologies available have strengths and weaknesses, depending on the application or process to be automated. Some example specialist solutions include the Hummingbird (Cartesian), the Echo 550 (Labcyte) and the mosquito (TTP LabTech).
 
The Hummingbird is a low-cost, high-speed instrument using glass capillaries in 96- or 384-well format, commonly used for plate-plate copies or reformatting in nanoliter volumes. It offers simultaneous transfers in the 25-1000 nL range and non-contact dispensing. Extensive washing is required between transfers to prevent cross-contamination and serial dilutions are not possible. For cells, Cartesian instead recommend their synQUAD system.
 
The Echo 550 uses bursts of focussed acoustic energy to propel nanoliter DMSO-based droplets between opposed microplates. It offers zero-cross contamination and hit-picking capability, and can prepare independent compound dilutions by volumetrically dispensing several different stock concentrations. However, the Echo 550 requires a large capital expenditure, is limited in its dynamic range (2.5-250 nL), and its accuracy is only guaranteed for solutions that are DMSO-based.
 
Where flexibility is required at a manageable cost, mosquito provides plate reformatting, copying, serial dilutions and assay-ready plate preparation the 50-1,200 nL range. Mosquito guarantees zero cross-contamination by using a continuous reel of low-cost disposable micropipettes. These use positive displacement, ensuring accuracy with different solution viscosities, and can aspirate, dispense and mix. They are also robust enough to pierce plate-seals. The pipettes' column arrangement allow mosquito to perform traditional serial dilutions within flat, round-, and V-bottom microplates. Importantly, it has been validated to pipette cells into 1536-well plates using a high content cytotoxicity assay.
 
 
Automating assay miniaturization
 
There are now multiple solutions available to move existing HCS into higher density miniaturized formats. These can be divided into three main approaches.
 
The first is to install an off-the-shelf uHTS system offering 24/7 unattended operation. Examples include systems developed by Kalypsys, Aurora Discovery and Evotec Technologies, which allow rapid implementation but at considerable capital investment. These complete solutions store and configure compounds, perform assays, manage the large amounts of information gathered and free scientists from repetitive tasks. Low-volume pipetting operations are performed using pin-tools, solenoid or piezo-electric actuated pipette heads, which require wash steps between transfers.
 
A second approach is to divide the process into modules: compound storage, cell culture, plate production and high-content readers may be integrated into a compact working space, like Protedyne's BioCube and Velocity 11's Biocel. This means capacity is easily scaled and tailored technology, for example, for nanoliter compound handling, may be integrated into the system.
 
Finally, the most flexible approach is to select instrumentation from a variety of sources and integrate it using appropriate robotics. This can be the most cost-effective method for those who simply require a specialist instrument to ease a bottleneck and fit with existing laboratory equipment.
 
When deciding on the best fit, key criteria include cost, throughput, integration with existing laboratory equipment, compatibility with the assay biology and the types of reagents to be pipetted.
 
 
Conclusions
 
The benefits of assay miniaturization in cell-based assays are clear; however, selecting appropriate instrumentation is only one of the many challenges that must be overcome. Success requires a well thought out implementation strategy, the cooperation of multiple teams and open collaboration with technology providers. With a resolve on all parties to succeed, the adoption of low-volume, high-content assays as primary screens can allow more rapid and efficient generation of primary hits against emerging therapeutics targets.
 
 
Dr. Wayne Bowen is CSO at TTP LabTech. He received his doctorate in receptor biochemistry from the University of Glasgow, and subsequently worked in neuroscience at SmithKline Beecham. Sarah L Payne is an applications specialist and Ben J Schenker is a product manager for TTP LabTech.

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