Stacker Modules Used in a High-Capacity Robotics System for High Throughput Screening Compound Replication

High throughput screening is now established as a key component of the pharmaceutical lead identification process in many pharmaceutical companies. Over recent years, thanks to advances in assay technology, process automation, and logistics control, the throughput capacity of HTS groups has increased significantly. It is now entirely possible to screen corporate compound collections against an individual pharmacological target within a timescale of several weeks. Despite these improvements, many HTS groups find that their capacity is limited by the rate at which they can provide test compounds in a "screen-ready" format. This limitation is usually imposed by the capacity and productivity of the single-armed robotic systems utilized. We have recently constructed a robotic system aimed at overcoming this particular problem. This system uses purpose-built microplate stacker units that provide high-capacity microplate storage and, importantly, provide an easy and fast interface between the robotic system and the human operators. This paper describes this automation project and the benefits that have resulted from its deployment.     

Microbiological high throughput screening: an opportunity for the lead discovery process

Microbial HTS has been implemented at Rh?ne-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.     

High-throughput siRNA-based functional target validation

The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.     

The future of high-throughput screening

High-throughput screening (HTS) is a well-established process in lead discovery for pharma and biotech companies and is now also being set up for basic and applied research in academia and some research hospitals. Since its first advent in the early to mid-1990s, the field of HTS has seen not only a continuous change in technology and processes but also an adaptation to various needs in lead discovery. HTS has now evolved into a quite mature discipline of modern drug discovery. Whereas in previous years, much emphasis has been put toward a steady increase in capacity ("quantitative increase") via various strategies in the fields of automation and miniaturization, the past years have seen a steady shift toward higher content and quality ("quality increase") for these biological test systems. Today, many experts in the field see HTS at the crossroads with the need to decide either toward further increase in throughput or more focus toward relevance of biological data. In this article, the authors describe the development of HTS over the past decade and point out their own ideas for future directions of HTS in biomedical research. They predict that the trend toward further miniaturization will slow down with the implementation of 384-well, 1536-well, and 384 low-volume-well plates. The authors predict that, ultimately, each hit-finding strategy will be much more project related, tailor-made, and better integrated into the broader drug discovery efforts.     

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.     

High-performance liquid chromatographic–electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening

Within the pharmaceutical industry, significant resources have been applied to the identification of new drug compound leads through the use of high-throughput screening (HTS). To meet the demand for rapid analytical characterization of biologically active samples identified by HTS, the technique of high-performance liquid chromatography–electrospray ionization mass spectrometry (HPLC–ESI-MS) has been utilized, and the application of this technique specifically for the integration of natural product sample mixtures into modern HTS is reviewed. The high resolution provided by reversed-phase HPLC coupled with the gentle and relatively universal ionization facilitated by the electrospray process has had significant impact upon a variety of procedures associated with the HTS of natural products, including extract sample diversity evaluation, dereplication, structure elucidation, preparative isolation, and affinity-based biological activity evaluation.     

Implementing multilevel dynamic scheduling for a highly flexible 5-rail high throughput screening system

As automation solves the bottleneck involved in drug screening, new bottlenecks present themselves. Some of these bottlenecks include sample management, hit picking and confirmation, and reagents lost as a result of incomplete runs. To keep up with the demands of a large HTS department, scientists spend a disproportionate amount of time simply feeding these systems with samples and reagents. Automating the sample management functions directly on the screening systems would solve this problem. With the use of online data analysis, an integrated sample store permits automated hit picking and confirmation. In addition to these issues, other bottlenecks are often caused by instrument malfunctions. A single lost run can now mean a loss of hundreds of plates and the reagents associated with their testing. A system was designed to include four assaying systems that are fed by an automated online sample repository system. Redundancy between the assaying systems allows for an extra level of error handling in case of a malfunction. The control of such a system requires a sophisticated scheduler/controller software package capable of coordinating the interaction between multiple systems and reacting to changes in the robotic environment in realtime. This paper discusses the design of the system as well as the requirements and selection of an appropriate scheduler/controller package.     

Standardization of automated cell-based protocols for toxicity testing of biomaterials

Advances in high-throughput screening (HTS) instrumentation have led to enormous reduction of costs (e.g., of pipetting stations) and to the development of smaller instruments for automation of day-to-day routines in small research laboratories. In the biomaterials community, there has been an increasing interest for standardized screening protocols to identify cell type-specific cytocompatible biomaterials suitable for tissue engineering (TE) applications. In this study, the authors established a multiplexed assay protocol for toxicity screening of biomaterials using a low- to medium-throughput robotic liquid handling station (LHS). The protocol contains analysis of viability, cytotoxicity, and apoptosis combined in one assay. This study includes performance results of a side-by-side comparison of the EpMotion 5070 LHS and conventional pipetting/dispensing systems. Critical parameters were optimized each for a given platform. Higher accuracy and reproducibility were achieved for LHS compared to manually treated samples. The practicability and accuracy of the method in a typical small laboratory setting were tested by running daily routine tasks by trained and untrained laboratory staff. In addition, advantages and disadvantages as well as the step-by-step application protocol are reported. The approach described provides a potential utility in screening biomaterials toxicity, allowing researchers to meet the needs of low- and medium-throughput laboratories.     

Automated assay optimization with integrated statistics and smart robotics

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.     

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-μmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.     

High-throughput screening: update on practices and success

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.     

The future of laboratory automation.

Among the many factors that will define the laboratory of the future are the development of advanced computer communications systems, artificial intelligence, robotic systems, and material storage and retrieval systems. This article examines some of these factors and challenges current automation justification procedures in light of the greater competitive environment of today.     

Identifying actives from HTS data sets: practical approaches for the selection of an appropriate HTS data-processing method and quality control review

High-throughput screening (HTS) has achieved a dominant role in drug discovery over the past 2 decades. The goal of HTS is to identify active compounds (hits) by screening large numbers of diverse chemical compounds against selected targets and/or cellular phenotypes. The HTS process consists of multiple automated steps involving compound handling, liquid transfers, and assay signal capture, all of which unavoidably contribute to systematic variation in the screening data. The challenge is to distinguish biologically active compounds from assay variability. Traditional plate controls-based and non-controls-based statistical methods have been widely used for HTS data processing and active identification by both the pharmaceutical industry and academic sectors. More recently, improved robust statistical methods have been introduced, reducing the impact of systematic row/column effects in HTS data. To apply such robust methods effectively and properly, we need to understand their necessity and functionality. Data from 6 HTS case histories are presented to illustrate that robust statistical methods may sometimes be misleading and can result in more, rather than less, false positives or false negatives. In practice, no single method is the best hit detection method for every HTS data set. However, to aid the selection of the most appropriate HTS data-processing and active identification methods, the authors developed a 3-step statistical decision methodology. Step 1 is to determine the most appropriate HTS data-processing method and establish criteria for quality control review and active identification from 3-day assay signal window and DMSO validation tests. Step 2 is to perform a multilevel statistical and graphical review of the screening data to exclude data that fall outside the quality control criteria. Step 3 is to apply the established active criterion to the quality-assured data to identify the active compounds.     

Strategy for screening metagenomic resources for exocellulase activity using a robotic,high-throughput screening system

Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11–14 h at 37 °C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-β-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10⁴ clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.     

A lightweight,inexpensive robotic system for insect vision

Designing hardware for miniaturized robotics which mimics the capabilities of flying insects is of interest, because they share similar constraints (i.e. small size, low weight, and low energy consumption). Research in this area aims to enable robots with similarly efficient flight and cognitive abilities. Visual processing is important to flying insects' impressive flight capabilities, but currently, embodiment of insect-like visual systems is limited by the hardware systems available. Suitable hardware is either prohibitively expensive, difficult to reproduce, cannot accurately simulate insect vision characteristics, and/or is too heavy for small robotic platforms. These limitations hamper the development of platforms for embodiment which in turn hampers the progress on understanding of how biological systems fundamentally work. To address this gap, this paper proposes an inexpensive, lightweight robotic system for modelling insect vision. The system is mounted and tested on a robotic platform for mobile applications, and then the camera and insect vision models are evaluated. We analyse the potential of the system for use in embodiment of higher-level visual processes (i.e. motion detection) and also for development of navigation based on vision for robotics in general. Optic flow from sample camera data is calculated and compared to a perfect, simulated bee world showing an excellent resemblance.     

Comparison of high throughput screening technologies for luminescence cell-based reporter screens

As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.     

Quantitative Progress Evaluation of Post-stroke Patients Using a Novel Bimanual Cable-driven Robot

Rehabilitation is the most effective way to reduce motor impairments in post-stroke patients.This process demands several hours with a specialized therapist.Given resources and personnel shortages,the literature reports a high interest in robotic assisted rehabilitation solutions.Recently,cable-driven robotic architectures are attracting significant research interest for post-stroke rehabilitation.However,the existing cable-driven robots are mostly unilateral devices allowing the rehabilitation only of the most affected limb.This leaves unaddressed the rehabilitation of bimanual activities,which are predominant within the common Activities of Daily Living(ADL).Thus,this paper presents a specific novel design to achieve bimanual rehabilitation tasks that has been named as BiCAR robot.Specifically,this paper provides a full insight on the BiCAR sys-tem as well as on its dedicated developed software BiEval.In particular,BiEval software has been developed as based on a serious game strategy and a virtual reality environment to track the patient exercising duration,motion ranges,speeds,and forces over time for achieving a quantitative assessment of the rehabilitation progress.Finally,the paper presents the BiCAR/BiEval capabilities by referring to a pilot Randomized Controlled Trial(RCT).The clinical trials have been used to validate the BiCAR/BiEval in terms of engineering feasibility and user acceptance to achieve an innovative cost-oriented integrated hardware/software device for the bimanual assistive rehabilitation of post-stroke patients.     

An effective tool for homogeneous assays using a simple robot arm with Stackers-TRISTAN

The Twister & RapidPlate Integrated System by TANabe, or TRISTAN, consists of a 96-channel dispenser (RapidPlate 96), a plate reader (V-MAX), and a simple robot arm (Twister). We developed TRISTAN for effectively conducting a homogeneous assay. Although this system accommodates fewer than 20 microplates, it has several advantages over conventional robotic systems for high-throughput screening in the following aspects: parameter setting, running time, hardware errors, manpower, and cost-effectiveness. The system proved to be effective and efficient for homogeneous assays.     

The design and development of an integrated natural products screening database

We designed and developed NEXUS--a new natural products screening database and related suite of software applications--to utilize the spectacular increases in assay capacity of the modern high throughput screening (HTS) environment. NEXUS not only supports seamless integration with separate HTS systems, but supports user-customized integration with external laboratory automation, particularly sample preparation systems. Designed and developed based on a detailed process model for natural products drug discovery, NEXUS comprises two integrated parts: (1) a single schema of Oracle tables and callable procedures and functions, and (2) software "front-ends" to the database developed using Microsoft Excel and Oracle Discovery/2000. Many of the back-end processing functions were written in Programming Language/Structured Query Language (PL/SQL) to provide an Application Programmer's Interface, which allows end users to create custom applications with little input from information technology professionals.     

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.