How do we know if a new drug is effective and safe? How to determine the mechanism of action of a new pharmaceutical compound? All these questions, and more, are made possible through in vitro cell viability studies.
Cell viability studies are a major component of modern drug development. They assess the toxicity and potential therapeutic effects of a new drug candidate by measuring the proportion of living cells within a treated cell population. They also determine how cells survive upon exposure to a compound, thus playing a key role in identifying safe and effective drug leads during preclinical stages of drug discovery.
Types of Cell Viability Assays
- Dye exclusion method – It differentiates between live and damaged or dead cells by staining with a dye (viz., Alamar Blue, trypan blue, Congo red, eosin, and propidium iodide) as the dye only penetrates damaged cell membranes.
- WST‐1 Cell Cytotoxicity Assay is a sensitive and accurate assay for cell cytotoxicity and proliferation. The assay is highly convenient as it is performed in a single tissue culture well and requires no cell washing, harvesting, or solubilization. Adherent or suspension cells are cultured in a microplate and then incubated with WST‐ 1, and the assay is monitored with a spectrophotometer. The assay principle is based upon reducing the tetrazolium salt WST‐1 to formazan by cellular dehydrogenases. The generation of the dark yellow colored formazan is measured at 420‐480nm (optimal at 440nm) and is directly correlated to cell number.
- MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay – MTT (a simple and sensitive assay) determines the mitochondrial activity by measuring the rate of conversion of yellow tetrazolium salt to a purple formazan product by mitochondrial dehydrogenase. The rate of conversion is directly proportional to the number of viable cells. However, the limitation consists of temperature dependency (a follow-up step of incubation of 1-4 hours) and pH (affecting absorbance). Thus, not practically applicable for all cell types.
- Sulforhodamine B (SRB) assay - a widely used cytotoxicity assay, employs the binding of SRB, a bright pink, fluorescent dye, to the basic amino acids of cellular proteins in a slightly acidic environment and its dissociation in a basic environment. Compared to other assays, it is independent of metabolic activity and exhibits low interference. CytoScan SRB Cytotoxic assay offer by G-Biosciences is based on SRB agent
- ATP assay: This is used to assess cell viability by measuring the ATP activity in the cell either by fluorescence, bioluminescence, or calorimetry. It is highly sensitive and can provide results in real time; however, the presence of ATP-degrading enzymes in the sample can jeopardize the results. G-Biosciences Lumino™ Cell Viability Glow Assay is a bioluminescent assay to detect and/or quantify ATP and thus measures the viable cells.
- Colorimetric cell survival assay: A reliable colorimetric assay that measures the stable, cytosolic, lactate dehydrogenase (LDH) enzyme, which is released from damaged cells and is a biomarker for cellular cytotoxicity and cytolysis. The released LDH is measured with a coupled enzymatic reaction that results in the conversion of a tetrazolium salt (iodonitrotetrazolium (INT)) into a red color formazan by diaphorase. The LDH activity is determined as NADH oxidation or INT reduction over a defined time period. The resulting formazan absorbs maximally at 492nm and can be measured quantitatively at 490nm. G-Biosciences offers the CytoScan™ LDH Cytotoxicity Assay Kit, which is used with different cell types for assaying cell-mediated cytotoxicity and cytotoxicity mediated by chemicals and other test compounds.
- Protease viability marker assay: This assay measures live–cell protease activity by using a fluorogenic substrate (GF-AFC), which is cleaved by proteases in live cells. A fluorescent signal proportional to the number of viable cells is generated. It offers less incubation time (0.5-1 hour) than MTT, does not depend on cell lysis, and is non-toxic. However, it exhibits sensitivity to the presence of protease inhibitors.
- Clonogenic cell survival assay: This assay assesses the reproductive viability of a single cell to form colonies. It quantifies cell survival after treatment with drug/cytotoxic agents and exposure to radiation.
- DNA synthesis cell proliferation assay –It measures the rate of DNA synthesis and can be used by employing BrdU (a thymidine analog, 5-bromo-2′-deoxy-uridine) and 3H-thymine (a radioisotope), which incorporates into DNA. EdU (a thymidine nucleoside analog, 5-ethynyl-2′-deoxyuridine) detects DNA synthesis without denaturing it using click chemistry. This assay is highly sensitive, expensive, and time-consuming. Additionally, it entails the use of radioactive materials.
With the extensive availability of cell viability assays, the ideal choice depends on the dynamic research question, measurement correlation with cell viability, types of cells, culture conditions, and
and limitations of the assays. G-Biosciences offer different cell viability, apoptosis assays & accessories.
Summary
Cell-based approaches are indispensable in modern drug development. For instance, they are used to assess the viability of cells in the presence of a particular drug, providing the safety, efficacy, and cytotoxicity parameters of candidate drugs. They can also be used to optimize drug formulations and predict possible drug interactions and any adverse effects. Secondly, cell viability studies can be automated, reducing time and allowing for high-throughput screening of large compound libraries. Therefore, this accelerates the identification of potential drug candidates. Also, these approaches offer more relevancy and accuracy when dealing with human cell lines than in-vivo studies. The effect of the drugs on human physiology and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties can be predicted more accurately. These assays offer reliability, reproducibility, and cost-effectiveness during drug development and discovery.
Despite being the need of the hour and providing various advantages, cell viability assays still have some lacunae. They lack the ability to encapsulate the complexity of biological systems, fail to consider some pharmacokinetic factors at play, are time-consuming, are cytotoxic, have cell counting errors, are difficult to process large datasets, and exhibit fluorescence interference.
To overcome these limitations, it is better to rely on a consistent source of cells and accompany the cell viability assay with cell cytotoxicity assays. This overcomes its dependency on fluorescent accumulation, as cell viability decreases with longer periods of incubation.
Figure: CytoScan™ WST-1 Cell Proliferation Assay
References:
- Liu, Bi-Yu et al (2022) CHINESE J PHYSIOL. https://doi.org/10.4103/0304-4920.354803
- Hayek, Hassan et al (2021) J BIOCHEM. https://doi.org/10.1016/j.jbc.2021.100578
- Lin, Y. et al (2017) Mol Immunol. 83:82
- Chow, Zeta et al (2021) CELLS. https://doi.org/10.3390/ cells10051261
- Douida, A et al (2020) J Cell Mol Med. doi.org/10.1111/jcmm.15323
- Pereira, FP, et al (2020) Nat Comm. doi.org/10.1038/s41467-020-18928-1
