September 15, 2015 (Vol. 35, No. 16)

Cell-Based Assay Platforms are Evolving to Meet Diverse Challenges

Cell-based assay platforms are evolving to meet diverse challenges—mimicking disease states, preserving signaling pathways, modeling drug responses, and recreating environments conducive to tissue development.

GEN recently interviewed a number of experts on cell-based assay technology to get a sense of the state of the art and to find out where this technology might be most valuable to life sciences research.

GEN: What are some of the main challenges that are faced when validating cell-based assays?

Dr. Kelly: Considerable challenges come from using systems involving a living organism in the validation of cell-based assays. The characteristics of such systems will likely affect the criteria for validation suitability. These criteria might be specific for primary cells, immortalized cell lines, cancerous cell lines, or cells generated de novo from multipotent stem cells.

Chemical reagents are generally well characterized by parameters such as molecular weight, solubility, etc., which are unlikely to change between assays.

However, characteristics of primary cells or established cell lines, such as viability, growth phase, proliferation rate, level of metabolism, and even cell size are much more vulnerable.

Mr. Trinquet: Beyond developing the right cell-based assay, the main challenge remains the relevancy of the cell model for the target being investigated. Generally, a single assay must also be compatible with a broad variety of cell technologies/models, from engineered cells to more complex models, such as 2D, 3D, microtissue, primary culture, and induced pluripotent stem cell models.

This certainly adds some difficulty, given that protein expression levels may differ from one model to another. Also, these assays must generally translate well all along the value chain, from high-throughput screening to late stages of lead op, so that end users do not have to switch between too many assay technologies.

Dr. Hsu: Cell-based assays provide more biologically relevant information than biochemical assays for high-throughput screening and ADME/Tox. One challenge in developing and validating cell-based assays is to generate cells that reliably express the drug target and give reproducible results with good Z′ over time.

We developed and launched the industry’s first cell-based assays and profiling services for G-protein-coupled receptors. The expression of G-protein-coupled receptors has been worked out, but ion channels are challenging. Another challenge is to make sure the assays and readouts are target specific and predictive, with a good dynamic range and signal-to-noise ratio to differentiate compounds with different potencies and efficacies.

Dr. Khimani: Cell-based assays provide a complex and physiologically relevant medium to evaluate the effect of novel therapeutic or modulatory candidates. However, unlike traditional assay formats, cell-based assays introduce a number of challenging factors that must be considered—such as cell type, expression level, stability, and passage viability—when optimizing the assay conditions.

In addition, with complex cell-based assay systems, data extraction and signal-to-noise optimization can be time-consuming bottlenecks. Other challenges, particularly with high-content screening, include separate investments in instrumentation, training, data analysis, and data management, all leading to a lower throughput.

Dr. Fan: Cell-based assays are model systems, and the most critical challenge facing such assays is how well they reflect real biology. Cell-based assays offer great advantages over biochemical assays because they are conducted in cellular contexts. That said, most of the current cell-based assays use a homogeneous population of cells grown from immortalized cell lines, many of which express target proteins or reporters in excessive, nonphysiological amounts via transient transfection or randomly integrated stable clones. These cell models are far from the actual cellular context in normal or diseased tissue such as a tumor.

In addition, phenotypical consequences of an analyte of interest to the cell could reflect a combination of effects that a single cell-based assay would not be able to fully address. These factors impact the validation or correlation of the results of a cell-based assay with a phenotypical consequence, an animal model study, or a clinically relevant finding.

Dr. Piper: The most formidable challenge in generating and validating cell-based assays is achieving predictability and translatability. Next-generation re-targeting systems (such as the Jump-In™ platform) have made over-expression of genes, even multigene cassettes, fast, reliable, and easy compared to traditional single-cell cloning.

While simple overexpression of a target may be sufficient to drive a primary screen and identify hits, it often lacks a sufficiently complex pathophysiological context to robustly convert hits to lead candidates that are meaningful in clinical trials. These systems have value at early stages, but they would benefit from improvements or secondary screens that can better translate to clinical results.

Dr. Payne: The choice of a cell system remains a challenge. Cell lines produce reproducible results, but do not accurately model living systems. Although primary cells are more physiologically relevant, they are inherently variable, making it harder to deliver a robust cell-based assay.

Choosing appropriate endpoints can be time consuming: measuring one parameter is not enough to accurately determine the functionality of a drug. The ability to analyze several markers in multiplex assays provides greater information on drug efficacy and toxicity, the latter being important for failing flawed drugs earlier. Finally, once validated offline, assays still require revalidation when transferred to automated contexts.

GEN: Is there new technology currently available or on the horizon that you feel will have a big impact on cell-based assays?

Dr. Kelly: Rapid advances in 3D cell-culture technologies enable creation of tissue-like structures in vitro. Traditional cell-culture methods do not address the complexities that cells encounter in real-life tissues. 3D cell cultures better represent the behavior of cells by allowing them to interact with adjacent cells and maintain their normal 3D structure with minimal exogenous interference. The success of this technology will depend on the ability to validate these models, which will lead to wider adoption by the research community.

Mr. Trinquet: Major breakthroughs have already occurred with the emergence and improvement of technologies such as high-content screening. Again, the use of complex cell models (more and more common in high-throughput screening), reconstituted microorgans, and induced pluripotent stem cells will obviously further relevancy, while new genome-editing approaches will also allow us to expand the variety and the accessibility of cell models.

Dr. Hsu: Currently, major types of cell-based assays used in high-throughput screening are second-messenger, reporter, cell-proliferation/cytotoxicity, and biosensor assays; most use overexpressed cell lines on a 2D surface. Recent progress has been made in cell culture tools mimicking the native environment, such as 3D cultures, microfluidic devices, and perfusion cell cultures using disease-relevant primary cells rather than established cell lines. Coupled with nondisruptive detection technologies, such as carrier-free nanoprobes and label-free technologies, these tools provide the ability to monitor endogenous targets in physiologically relevant environment in single live cells.

Dr. Khimani: The expansion of 3D cell models and associated assay systems will provide a significant impact on cell-based assays in the near future. In these models, the architecture closely mimics real biological environments, such as those characterized by cell–cell interactions, and has the complexity to acquire high-content, multiparametric data from a single assay.

Another area to revolutionize cell assays is single-cell analysis, both at the genomic and proteomic levels. Single to several cells per well will enable multiparametric data resolution and facilitate throughput via miniaturized formats.

Dr. Fan: We are at an exciting crossroads where several new technologies will revolutionize cell-based assays to overcome current limitations. Genome-editing tools such as CRISPR/Cas9 will allow easy engineering of mutations, knock-outs or knock-ins of a specific reporter or marker at precise location on the genome. 3D culture models, co-culture of cells, and artificial tissue techniques move us closer to a cellular context.

Single-cell isolation, advances in super-resolution imaging, and next-generation sequencing enable analysis at the level of an individual cell as opposed to a large population of cells. Conclusions of big data generated from a single cell should lead to new or better insight into many disease mechanisms.

Dr. Piper: Primary cells or reprogrammed induced pluripotent stem cells from donors with known genotypes and clinical symptomology promise more direct translation of in vitro results to clinical outcomes. Use of genome-engineering technologies (such as TALEN and CRISPR/Cas9 systems) will be used to generate isogenic disease model platforms and gene knock-down screens that focus on the underlying genetic, molecular, and cellular mechanisms of the specific disease. Moreover, these models, coupled with multiple detection technologies to study orthogonal cellular endpoints (such as Ca2+ handling and phosphorylation of a target protein), will offer even greater utility and predictability.

Dr. Payne: The emergence of synthetic biology tools and circuits are redefining the future of cell-based assays within many drug discovery stages. Recent progress in DNA manipulation and genetic engineering has improved the ability to program and probe mammalian cell behavior, providing novel research tools for the elucidation of disease mechanisms and target identification. It has also revealed potential opportunities for the development of innovative biotherapeutics (cell-based therapies, protein drugs, vaccines, and gene therapies).

GEN: What is more valuable to researchers with respect to cell-based assays miniaturization or ultra-high throughput?

Dr. Kelly: A single cell contains the complete genome of the species and thousands of expressed genes, implying that one cell could provide the same information as millions. High-throughput efforts should be aimed at our ability to multiplex, multivisualize, and microarray the enormous amount of information that one cell can provide.

Mr. Trinquet: Miniaturization may be more important because the cells that are used are more complex and costly to produce massively. It comes to be particularly important when several assays need to be run in parallel using the same sample, such as cell lysate after stimulation.

Dr. Hsu: To achieve “ultra-high throughput” rates in excess of 100,000 assays per day, the assays have to be miniaturized. The trend toward assay miniaturization requires development of homogeneous, fluorescence-based assays with higher sensitivity and throughput. Miniaturization and ultra-high throughput go hand in hand; they depend on each other.

Dr. Khimani: Ideally, a combination of miniaturization and ultra-high throughput would facilitate reagent cost savings as well as the screening of more drug candidates. However, biological relevance and data quality are priorities. Access to appropriate and reproducible cell models, optimized assays, signal-to-noise optimization, and accurate data analysis/management capabilities deliver significant value to the screening effort.

Dr. Fan: Cell-based assays are useful for a wide variety of applications. Trends such as miniaturization and ultra-high throughput have the potential to expand the uses of cell-based assays even further.

Dr. Piper: While either may help the speed at which researchers gain results, the key to innovating discovery will be added depth and quality of the data.

Both miniaturization and ultra-high throughput, in combination with advances in high-content imaging and flow-based analysis along with better cellular models, will be required to provide more relevant and meaningful data.

Dr. Payne: Trends to increase the physiological significance of screens using high-content assays and relevant cell models are offset by a negative impact on cost/test and throughput. Miniaturization decreases cost/well. However, for most high-content instruments, throughput is not significantly improved. Laser scanning cytometry provides an alternative approach for those who value both.