Drug Discovery

Since its inception in the mid 1990's, High Content Screening and analysis (HCS/A) technologies have become ever increasingly adopted within the fields of drug discovery and cell biology. One of the most attractive features of this technology is that it provides contextual information at the level of the intact cell or tissue and permits the quick, straightforward and reproducible measurement of the morphological and structural properties cells and organelles (Korn et al 2005; Giuliano K.A. et al 2005). HCSA technologies represent the convergence of several mature laboratory analysis technologies, namely fluorescent light microscopy coupled with the automation and ease of use of a plate reader and the ability to analyse cellular subpopulations functionality associated with flow cytometry.

The key feature of this technology is the ability to perform highly detailed analysis on cellular images acquired by these platforms. This combined functionality hence enables for the first time the study of the functional characteristics of genes and proteins in the context of the whole cell (a definition of the discipline cellomics). HCSA currently commands a major place in the drug discovery process as it provides researchers with massive amounts of biological information that is relevant to pathway analysis. With HCSA in the market growth stage, the ADVANCE 1536 nano well plate will take a significant place in the drug discovery process in the next 5 years. It will result in more accurate and better leads, in less time, with less reagent use. So that larger libraries can be screened, increasing the information content of the screen and increasing the odds of finding good leads.

To reduce costs, the pharmaceutical industry is attempting to improve efficiency by investing in automated technologies that utilise more physiologically relevant cell based models. An example of such a technological approach is the newly emerging High Content Imaging Technologies. High Content Imaging Technologies are becoming increasingly used within drug discovery as they permit a rapid and robust means of identification and validation of therapeutic as well as providing a means of assessing the efficacy and/or toxicity of drug candidates. Despite the obvious advantages this technology offers, the reagent costs associated with cell based screening can be prohibitive.

With the advent of micron resolution robotics and nano litre capable liquid handlers, large scale and automated assay miniaturisation is now possible. The advantages of miniaturisation are clear, when one considers the savings in reagents and experimental materials.

The growing trend toward biological assay miniaturisation has been driven by the need to reduce costs and has been facilitated by scientific and technological progress in automation and detection instrumentation. The best contemporary example of biological assay miniaturisation is the DNA microarray (Schena et al, 1995) and protein microarrays (MacBeath & Schreiber, 2000). These analytical tools provide a fast and inexpensive means of gathering highly detailed information on gene expression or protein interaction at the cell or tissue level.

Cell-based assays are commonly performed in micro-titre plates which are now available in a multitude of well densities ranging from 96-1536 wells / plate (with working reagent volumes from 5 to 100 µl per well depending on the plate format). Although micro-titre plates are ubiquitously used in cell biology labs, the experimental costs of performing large scale experiments using these technologies even in the high density formats can be prohibitive. As such, many are now turning to cellular micro array technologies as an alternative to the more costly conventional micro plate technologies.

In the conventional HCS assay preparation workflow these five discrete steps are followed:

1. Assay preparation involves setting up the cell based assay for the screen: This step is often preceded by an assay development step where the assay is optimised and assessed for use within the High Content Screen. As an example a typical assay workflow for high content screening is performed in three discreet workflows namely (i) cell seeding, Compound and (ii) ligand dosing and (iii) Fixation/Permeabilisation, washing, antibody and substrate addition.

2. Image acquisition involves the capture of high resolution digital images using automated multi-channel fluorescent microscopy e.g. INcell 1000. These systems are typically used in conjunction with multi-welled plates and are capable of automatically focusing, filter wheel, sample alignment (moving stage) image capture and data storage.

3. Image analysis initially begins with the selection and configuration of an image analysis algorithm which has been setup to selectively identify and segment targets of interest such as cells or sub cellular regions. The image analysis process may be subdivided into the following steps (i) image pre-processing to e.g. correct for uneven illumination, (ii) image segmentation in order to outline the objects of interest, (iii) feature extraction in order to quantify the characteristics of the objects of interest.

Figure 2. Example of object selection and subpopulation analysis where images of peripheral t-lymphocytes are analysed and selected on the basis of their gross morphology (i.e. the elongated cells (green) and round cells (red). (Images,courtesy of Dr Sowtheng Ong Institute of Clinical Medicine, Trinity College Dublin).

4. Data Analysis and Visualisation: Current methods are limited and typically provide only basic levels of information mainly comprising statistical outputs using Z-scores for identification of 'hits' (i.e. genes altered with respect to controls). Data Visualisation formats include (i) heat maps, (ii) correlation plots and (iii) plate-well scatter plots as a measure of the HCS performance and reproducibility.

Figure 3. Data obtained from HCS experiments using data visualisation tools (Birmingham et al Nat Methods. 2009 August; 6(8): 569575).

Figure 7. Phase contrast (upper) and fluorescence (lower) microscopy image of live, intact DRAQ5-stained U2-OS cells showing the differential two-compartment rendering of nuclei and cytoplasm permitting more robust tracking of protein re-distribution and translocation events and cell morphometrics.

Figure 8. Two-colour merged image of GFP-cyclin B (green) expressing U2-OS cells co-labeled with CyTRAK Orange (red) as nuclear counter stain. CyTRAK Orange is spectrally separated from GFP permitting imaging of the two separate signals.

Figure 9. Cells co-labelled with the mitochondrial membrane potential probe JC-1 which disaggregates on de-polarization with a chromophore change from orange to green. The far-red viability probe DRAQ7 (false-coloured in blue) was used to label cells with compromised membranes. The two cells in the upper panel exhibit intact membranes but different degrees of mitochondrial health while the cell in the lower panel exhibits both mitochondrial membrane potential and plasma membrane integrity collapse.

Figure 10. Acumen scan of two colour cell-based assay. Cells have been identified using cytometric analysis based on their higher fluorescence staining compared to the background.

During scanning, data is processed immediately upon acquisition by proprietary signal thresholding algorithms without the intermediate generation of an image file. This significantly reduces the amount of data generated and the requirement for large capacity data storage equipment (a prerequisite for microscope-based imagers). To date Acumen has not been promoted as an image capture device due its primary role as a high-speed system for screening large libraries of chemicals or genes (1, 2). However, it is possible to create images using the raw scan data which can be readily imported into third-party image analysis software. For this project, there is a requirement for generation of multicolour, high resolution images of whole assay wells. Laser scanning is the only viable approach while achieving high sensitivity and rapid sample throughput.

A second consideration is the availability of novel labware (e.g. plates and slides) that enable high content analysis of previously incompatible biological applications (e.g. chemotaxis, cell migration).

Figure 11. Whole well image of microtubule formation in an angiogenesis assay generated by Acumen. Inset: false coloured magnified section of the well.

Key Aspects of the ADVANCE1536 Nanowell Plate

The Missing Piece

The Missing Piece
Allowing researchers to
mimic in-vivo conditions

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Reduced Edge Effects

Reduced Edge Effects

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The Missing Piece

Market Drivers

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The Missing Piece

Miniaturisation

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