Cell Assays – Present & Future (Part Two)

Written by SelectScience Guest Editor, Dr Peter Simpson, AstraZeneca.

Assays form the backbone of the compound ’design-make-test-analyze’ cycle of data generation and decision-making in drug discovery projects. To enable good decision-making, assays have to be designed, built and prosecuted with skill and care. Assay scientists seek to build assays that reliably estimate the activity of compounds, while retaining relevance to the biological mechanism of the target under investigation. In the second part (part one can be read here) of this article on cell assays, liquid chromatography, mass spectrometry and 2D/3D cell culture technologies and methods are discussed.

The Use of LC-MS in Cell Assays
For many years, liquid chromatography - mass spectrometry (LC-MS) has been an important, but late, component of drug testing cascades. Mass spectrometry can be highly desirable as an assay, as it is a highly specific detection that can enable targets that are not amenable to most other assay types. For example, minor changes in a metabolite due to enzymatic activity of the target can be detected via mass spectrometry, a situation for which it would be impossible to generate a specific, selective antibody-based assay. In many cases, labeling of proteins is not required, and sensitivity can be sufficiently high for assays to be performed in native or primary cells. Cell LC/MS screening assays with detection of multiple endpoints in parallel are an attractive option; for example, metabolism targets in which multiple pathway components may be affected by a compound, and inhibition at one point can be counteracted by compensation elsewhere in the pathway or network. However, implementation of LC-MS in routine screening has been limited by assay complexity and technology throughput.

Technological advances in sensitivity and speed of commercial LC-MS systems, for example, RapidFire 360 High-throughput MS Systems (Agilent) and the simplification of the pre-processing of samples, has enabled processing speeds of under 10 seconds per sample for biochemical assays. Separation and quantitation of multiple parallel readouts from interfering cellular components in a timely way can now be used for complex metabolism drug targets. High performance UPLC systems such as the Waters Acquity I-Class are continuing to improve the ability of researchers to derive complex separations at increasing throughput. Acoustic injection may enable even higher throughput LC-MS in future. There are significant ongoing investments in sample preparation, acoustic injection and improved detection technology for mass spectrometry, so this is likely to be a growth area both in terms of throughput and sensitivity for assay screeners.

2D/3D Cell Culture Advances
Chemotaxis and cell migration monitor the movement of cells in 2D and have been adapted from low throughput experiments to higher throughput modalities. The advent of high content imaging has led to these assays becoming easier to measure in reasonable throughput.

Examples of this technology include the Platypus Technologies Oris™ Cell Migration and Invasion Assays, which provides spaces for migrating cells to fill using a preformed template, and the Bellbrook iuvo assay plate system, which utilizes a capillary for chemotaxis experiments. Quantitation of these assays ranges from simply measuring the size of area ‘closed’ or the distance migrated, to in-depth analysis of the movement of individual cells.

A fairly recent innovation in cell assays is the ability to measure cells growing in real time over prolonged periods. Bright-field imaging systems, such as IncuCyte™ Kinetic Live Cell Imaging System (Essen Instruments), now facilitate a range of experimental studies on long-term cellular behavior, as they can be left in the cell incubator for extended time periods. They are available with fluorescent options, enabling combined use of cell shape changes with fluorescent detection of biological events within the cell. Growth curves over many days, or tracking of apoptotic events for example, are particularly useful when looking at compounds that have more subtle growth inhibitory effects, or which may have a delay in cell toxicity or shape change, dependent upon their mechanism of action.

The above assays are designed to be used in traditional monolayers, or 2D cultures. Growth of cells in 3D cultures is claimed to more accurately recapitulate the in vivo environment, and has been the focus of much effort. Cells can be suspended in a semi-solid media comprising either agarose or cellular polymers, such as collagen, and grow as distinct clumps and clusters. Alternatively, a ‘hanging drop’ of growth medium can be used to encourage cells to cluster in interesting ways. There are coated plates available that allow for non-adherent 2D growth, said to more closely resemble 3D conditions.

The 3D nature of these approaches means that cells are more difficult to accurately visualize or detect for high content or biochemical analysis, although some assays to do this are improving. The difficulty of setting up robust routine 3D growth assays means that they are often currently reserved for projects where their need is absolute. These assays may, however, be a valuable option to use where there is good evidence for differential target or pathway expression in the 3D environment, compared to expression in conventional culture. Co-culture of multiple cell types that interact in vivo, such as cardiomyocytes and fibroblasts, may also encourage expression of more physiological cell phenotype and behaviors.

It remains the case that the most biologically relevant assays are relatively complex and more challenging to perform than the simpler, high signal-to-background assays that screeners have relied upon for years. There are new challenges for screeners, with the dawn of inducible pluripotent stem cells enabling relevant human biology in assays, and a drive to more complex (multicellular, three-dimensional) cultures.

A possible worry has been that the ability to interpret the biology of those systems will be constrained by previous reliance on simple detection assays. This can be addressed by combining the improved cell culture approaches (3D, human iPS-derived populations) with the types of assays described above that enable complex pathways and biology to be better understood.

Screeners will continue their transition to detection from relevant human cells, and to measuring multiple cellular events in parallel. This will be enabled by high content-based imaging, or mass spectrometry, or electrophysiological approaches, or other emerging solutions in label-free and biophysical detection. The ability to truly understand what is happening within a cell, in real time, and with multiple detections in parallel, will continue to fundamentally improve our ability to interpret how our compounds interact with proteins. This should facilitate development of improved lead molecules with more relevance to the in vivo situation, and therefore, in time, improved drug candidates.

[A longer article on current and future assays, by myself & Dr Tim Hammonds, will be published later this year in 'A Handbook of Medicinal Chemistry', RSC Books]