The implementation of cell-based assays can improve the process of drug discovery and development by providing important biological information about drug action mechanisms, off-target effects, and cellular viability and processes. The use of these assays can mitigate expensive efficacy and toxicity issues that may arise at the later stages of clinical trials.
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The international market for cell-based assays was estimated at $17 billion in 2016 and was expected to be worth $28 billion in 2021—with the US making up half of the market.
The primary causes of disability and mortality in the US are chronic diseases such as cancer, neurodegenerative disorders, cardiovascular disease, obesity, arthritis, and diabetes. The increase in disease burden, and the corresponding economic burden, has led to the growing use of cell-based assays.
The primary goal is to explore novel therapeutics or new methods of treatment and get them to the market in an effective and timely manner.
The data acquired from cell-based assays can also provide insight into disease mechanisms, improving scientific knowledge and discovering new therapeutic pathways and targets.
Types of cell-based assays
There are various types of cell-based assays, which can be designed to specifically address the demands of different researchers. The signal readout also varies and can be colorimetric, fluorescence, radioactivity, or luminescence, conditional on the variable being measured. Flow cytometers, microscopy, and plate readers can be used to measure signals.
High-throughput screening (HTS)
Cell-based assays are scalable and feasible and are thus being used in high-throughput screening (HTS) to concurrently analyze numerous compounds under a variety of conditions, yielding a large amount of data. This method is often employed to identify candidate drug leads at the start of the drug discovery process.
Cytotoxicity studies
After the identification of a lead candidate, the viability of healthy and diseased cells in the presence of the compound must be determined, ideally using a range of incubation times and concentrations. One of the most reliable ways to measure cell viability is to quantify intracellular ATP. A recent comparative study found that Promega’s CellTiterGlo reagent gave the greatest correlation with cell count data.
Cellular proliferation assays
Cellular proliferation is crucial to tissue and cellular equilibrium, which is needed for the proper growth, development, and maintenance of an organism. Cellular proliferation assays monitor the growth rate of cells over a given time and provide insight into how a drug will influence normal and diseased cells and tissue in vivo.
It is ideal if a compound inhibits cancerous cell growth while enabling the normal growth of healthy cells. Other cell-based assays can be used to probe the mechanism of growth inhibition.
Cellular death assays: Apoptosis, necrosis, and autophagy
By using dyes incorporated into dead or dying cells, the extent of induced cellular death in healthy or diseased cells can be determined. This can be translated into compound efficacy and potential toxicity in vitro. Insights into the mechanisms behind a drug’s actions can be gained by understanding how a given compound induces cell death.
The exposure of phosphatidylserine (PS) on the extracellular surface of the plasma membrane, mitochondrial membrane potential disruption, cell shrinkage, condensation, caspase activation, and DNA fragmentation are hallmarks of apoptosis (programmed cell death type 1).
A common measure of apoptosis uses conjugated annexin staining. Annexins bind to exposed PS in a calcium-dependent manner. The resulting signal (yielded from the conjugate) can be read by a plate reader or by flow cytometry.
Autophagy, or programmed cell death type 2, is the selective degradation of intracellular targets like damaged organelles and misfolded proteins. It plays a key role in the pathogenesis of numerous infectious and chronic diseases. Dyes can be utilized to measure autophagic vesicle accumulation.
Morphological changes (such as the destruction and swelling of the organelles and plasma membrane) are characteristic of necrosis (programmed cell death type 3).
Following innovations in technology and commercial kits, early apoptosis and subsequent neurosis can be measured in real-time using Promega’s RealTime-Glo Annexin V Apoptosis and Necrosis Assay, which provides detailed information about the drug’s mechanism of action.
Cellular signaling assays
Cell signaling assays identify how a candidate drug impacts cells in a paracrine and autocrine manner. Cell signaling can be measured using various methods, including changes in intracellular pH, proliferation, gene expression, metabolic assays, and intracellular calcium flux.
Signaling transduction cascades cause the modification or activation of downstream messengers. These changes, such as subcellular location or phosphorylation, can be analyzed by techniques like immunohistochemistry.
Metabolic assays
Changes in cellular metabolism are associated with neurodegeneration, cancer, and other diseases. Metabolic assays can offer important information into how a drug influences various aspects of oxidative stress and cellular metabolism.
For instance, oxygen consumption, enzyme activity, metabolite levels, glucose uptake, mitochondrial function, glycolysis, antioxidants, and fatty acid metabolism can be measured in response to drug therapy.
Receptor binding assays
Receptor binding assays can determine the ability of a drug to disrupt or promote an endogenous interaction between a ligand and receptor, the affinity of a drug-receptor interaction, and confirm receptor activation and inhibition. The activation of receptors be measured by assessing downstream events, like cyclic adenosine monophosphate (cAMP) generation for G protein–coupled receptors (GPCRs) or phosphorylation for tyrosine kinase receptors and calcium flux.
Gene reporter assay
Gene reporter assays deliver quick, robust, and sensitive signal outputs after the activation or inhibition of specific pathways, providing important mechanistic information about an early-phase drug. Primary gene responses, which can be timely to test and require a more sensitive approach, can then be validated.
Optimizing cell-based assays
One of the primary issues with cell-based assays is generating a consistent, reproducible assay that is reflective of a disease’s physiology and biology. After acquiring preliminary results, optimization is often necessary.
Data from one experiment often results in the generation of new assays, which provide additional knowledge about the potency of the drug and its mechanism of action. As a drug advances toward market release, better preclinical data will promote higher-quality clinical data.
Choosing an optimal cell/disease model
Most drugs fail to reach the market; thus, the drug discovery approach has been moving towards utilizing more relevant disease models. Primary cells and tissue obtained from diagnosed patients are therefore the best models for evaluating potential drug therapies.
A low passage number, consistent across all samples in the study, must be used. Immortalized cells have been shown to provide inconsistent results in cell-based assays and are not reflective of the disease biology.
Recent investigations have found that compared to immortalized Vero and Huh 7 cells, which require high virus input to spread and detect replication, primary macrophages are more susceptible to infection by Ebola. With increasing input virus and assay time, higher EC50s were also observed.
Vero cells showed substantial differences in the ability to be infected by the Ebola virus across passages—low and high passages were more refractory than cells at passage 10-12. The use of immortalized cells contributes toward non-reproducibility and non-clinical relevancy.
It has also been demonstrated that 3D cell culture models are more reflective of the genetic profiles of clinical specimens compared to conventional 2D culture models. 3D models may therefore be superior in predicting clinical efficiency, and may better match certain experimental designs (especially when evaluating tumor invasion assays or anti-tumorigenic compounds).
Good laboratory technical skills
Errors related to pipetting and seeding cells lead to inaccurate results, increasing variability within experiments. Volumes should be observed when pipetting and pipettes must be routinely calibrated. Dramatic differences in datasets can arise when diluting drugs, even from a few microliters in difference between samples.
This is particularly the case for multi-well pipettors, which often display variability in volume depending on how evenly or firmly tips are pressed into pipettors. To avoid the risk of cross-contamination, pipette tips should not be reused.
Researchers should be mindful to reduce the edge effect by utilizing specialized plates or allowing cells to sit at room temperature for a few minutes to permit even seeding before placing them inside an incubator.
Microplates must not be stacked atop each other, as this leads to uneven temperature and gas exchange, which causes variability across samples. The laboratory should be at room temperature when seeding cells, as it enables an even dispersal of cells across the wells.
Conducting strict aseptic techniques improves the quality of datasets, as bacterial, mycoplasma, or fungal contamination can significantly affect data and make valuable patient samples unusable.
Optimizing cell density and incubation time points
To determine optimal assay conditions, a pilot time-course experiment with multiple cell densities and multiple concentrations should be performed. Cells respond to drugs differently depending on their rate of growth, cell density, and incubation duration. The stability of a drug must be considered for longer periods of incubation—daily changes of media with fresh drugs could be beneficial.
A good understanding of the assay being used will help researchers to identify primary parameters. For instance, when measuring apoptosis, looking for early time points could be important as this event can be instigated rapidly.
Incorporating phenotypic and biochemical data
Phenotypical data can be utilized to validate biochemical data and can reveal unknown knowledge about a drug or disease system. Images of cells can deliver important information that raw numbers are unable to.
Having access to document images of cells during assay can help researchers find and explain potential issues that may arise since signal outputs are often measured by fluorescence and luminescence, which can be influenced by numerous variables.
About BioIVT
BioIVT, formerly BioreclamationIVT, is a leading global provider of high-quality biological specimens and value-added services. We specialize in control and disease state samples including human and animal tissues, cell products, blood, and other biofluids. Our unmatched portfolio of clinical specimens directly supports precision medicine research and the effort to improve patient outcomes by coupling comprehensive clinical data with donor samples.
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