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GENengnews.com be kept free of bacterial contamination. Such challenges have been addressed primarily with instrumentation and auto- mation. Incubators, which maintain en- vironmental conditions, can have built-in robotics to move microplates to and from liquid handlers and reader/imager devices to automate lengthy assay processes. Reagent additions or media exchanges are performed with liquid handlers, whereas detection (im- age based or quantitative) is carried out with a microplate reader. These systems are small enough to fit inside HEPA-filtered biosafety cabinets. Dr. Stump Cell-based assays enable one or more target-specific measurements amidst thousands of interdependent chemical reac- tions occurring inside each cell. The end goal is to identify a lead candidate based on a physiologically relevant response. To accom- plish this goal, assay design (including the choice of a model system, endpoints specific to the drug target, protocols, and reagents) must be robust. Thus, methods beyond 2D cell culture may be the answer, but they dras- tically increase the complexity of the assay. Adequate dynamic range of the assay is also a key challenge since nanomolar inhibi- tors require at least femtomolar detection for accurate curve generation. Highly spe- cific antibodies can address this issue by sub- stantially increasing the signal-to-noise raio. These and other elements combined must yield a reproducible assay that can achieve the throughputs desired. Dr. Charrier-Savournin Immortalized cell lines have been extensively used since they are cost effective and easy to manipulate ge- netically. However, they are not representa- tive of normal cells, so their use for screening assays becomes outdated. Closer to native pathophysiology are fully differentiated pri- mary cells, cancer cells, embryonic stem cells, or induced pluripotent stem cells, but they are less available, more difficult to maintain and differentiate, and more costly. Thus, cell-based assays need to adapt. They can become more sensitive, use less sam- ple, or even work on endogenous expression while keeping the characteristic advantages e while keeping the characteristic advantages eep while keeping the characteristic advantages g while keeping the characteristic advantages t while keeping the characteristic advantages e while keeping the characteristic advantages c while keeping the characteristic advantages a while keeping the characteristic advantages acte while keeping the characteristic advantages st while keeping the characteristic advantages c while keeping the characteristic advantages adva while keeping the characteristic advantages tages while keeping the characteristic advantages of high-throughput screening, which include robustness, ease of use, and miniaturization. Highly sensitive detection technologies, such as homogeneous time-resolved fluorescence energy transfer (TR-FRET) assays, have evolved to combine both requirements and are now fit to measure endogenous events in a screening environment. Ms. Gitschier Cell-based assays provide a biologically relevant environment, are ame- nable to high-throughput screening, and do not require the complexity and risk of whole- organism testing. A major challenge of cell- based assays involves choosing an ideal as- say to represent the system of interest, while maintaining consistency and reproducibility. Cell culture and plating techniques, the use of automation, and microplate selection can be optimized to reduce variability. Specifically for high-throughput screen- ing, evaporation and small-volume use are concerning. For these assays, microplate seals, low-evaporation lids, and specialty mi- croplates are useful. Finally, having vendors of instruments and microplates work closely together as technologies advance to ensure compatibility can help alleviate challenges customers face when adopting a new assay to meet their high-throughput drug discov- ery needs. Dr. Collins To more closely mimic disease, researchers are increasingly faced with the need to develop cell-based assays that accu- eed need to develop cell-based assays that accu- to need to develop cell-based assays that accu- deve need to develop cell-based assays that accu- op need to develop cell-based assays that accu- ce need to develop cell-based assays that accu- based need to develop cell-based assays that accu- assays need to develop cell-based assays that accu- t need to develop cell-based assays that accu- at need to develop cell-based assays that accu- accu need to develop cell-based assays that accu- rately reflect the environment of the human body. Scientists are studying 3D spheroids as a biologically relevant model that accurately replicates the in vivo environment. For ex- ample, variable aperture confocal technol- ogy on the IN Cell Analyzer 6000 is instru- mental in optimally imaging these challeng- ing samples. Another difficulty in developing better biological models is preparing samples in a miniaturized format. A common solution is round-bottom plates, which require a robust autofocus algorithm for automated imaging. Finally, studies of living biology are criti- cal to understanding dynamic cellular events. Recent speed improvements on the IN Cell Analyzer 6000 enable acquisition of beating cardiomyocytes at 42 frames per second, accu- rately representing cardiac physiology. D r. M o h a n Most high- throughput screening (HTS) in drug development is based on cell-based and biochemi- cal assays. Although cell- based assays are useful for target validation and AD- MET, they are not free from challenges. One of the great- est challenges is to develop stable cell lines that express reasonable quantities of target proteins. Another challenge is the decline in the expression level with each passage, which adds difficulty in data interpretation. Immortalized cell lines, such as HEK293 and cancer cell lines, are widely used, but they can harbor mutations. They may not give true results, that is, results consistent with those obtained with primary native cells. Some of the challenges can be over- come by the use of 3D cell cultures, which emulate in vivo morphology as well as cell- cell and cell-extracellular matrix interac- tions. Dr. Chandy Some of the challenges in using cell-based assays are choosing a cell model that allows throughput and standardizing image analysis and interpretation for com- age image analysis and interpretation for com- a image analysis and interpretation for com- a image analysis and interpretation for com- ys image analysis and interpretation for com- s image analysis and interpretation for com- a image analysis and interpretation for com- d image analysis and interpretation for com- te image analysis and interpretation for com- p image analysis and interpretation for com- etat image analysis and interpretation for com- o image analysis and interpretation for com- o image analysis and interpretation for com- co image analysis and interpretation for com- plex assays, such as time-lapse or 3D objects. In particular, imaging of 3D models may bet- ter predict reactions of or more closely mim- ic the in vivo environment. Previously, using live cell assays where 3D acquisition was required forced researchers to compromise between resolution and phototoxicity during z-sectioning. Modern systems, such as the Imag- eXpress ® Micro Confocal system, enable high-resolution screening at the speed of f high-resolution screening at the speed of widefield imaging through advanced illu- mination techniques and optics. Integrated software packages, such as MetaXpress software, allow for multiple cell types, sub- strates, or cell behaviors to be automatically characterized at once, reducing variability across experiments. Dr. Khimani Although cell-based assays of- fer physiologically relevant systems for drug discovery, they traditionally provide output in just one dimension. Such output may miss effects such as molecular, morphological, and other phenotypic changes that could challenge further development of a drug can- didate. Instead, cell-based strategies including multiparametric and multianalyte readouts at the subpopulation- or the single-cell level should be considered. Multimode and multi- analyte analysis tools that detect changes in a spatial context are critical in determining both the efficacy and toxicity of a drug candi- date as well as better downstream outcomes. Furthermore, current advances and contin- ued development and integration of 3D cel- lular models, simultaneous detection, imag- ing, and data analysis capabilities will further enable high-content drug discovery. Dr. Riss In general, the biology of the high- throughput assay system contributes more to variability than the chemistry of the detec- tion reagents. Perhaps the biggest challenge for cell-based high-throughput screening is having uniform samples of cells in as- say wells at the time the measurements are made. Variability in dispensing and culturing small numbers of cells in low-volume plates often leads to edge effects that result in vari- ab ability among replicate samples. ty ability among replicate samples. a ability among replicate samples. o ability among replicate samples. g ability among replicate samples. ep ability among replicate samples. cate ability among replicate samples. sa ability among replicate samples. p ability among replicate samples. es. ability among replicate samples. Roundup Continued from page 12 DRUG DISCOVERY E X P E R T P A N E L Chandra Mohan, Ph.D. Senior Manager Scientific Content and Training MilliporeSigma Grischa Chandy, Ph.D. Senior Product Marketing Manager Molecular Devices Anis H. Khimani, Ph.D. Head of Strategy and Marketing, Research Reagent Solutions PerkinElmer Terry Riss, Ph.D. Global Strategic Marketing Manager Cell Health Promega Robert Horton, Ph.D. Product Manager Discovery Assays and Services Thermo Fisher Scientific 14 | AUGUST 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Cell-based assays provide the complexity required for drug-based responses, offer mechanistic insights, and are amenable to faster assay times. They have great potential for producing patient-centric data.