The Evolution Of Cck-8 Assays In Cell Viability Analysis

Cell-based assays are widely used to assess compound-induced cell proliferation and cytotoxicity, aiding in the identification of molecules that either enhance growth or induce cell death. These assays also facilitate the study of receptor activity and signal transduction, including gene expression, intracellular trafficking, and organelle function.

The Cell Counting Kit-8 (CCK-8) assay, a sensitive and non-toxic colorimetric method, is frequently employed to quantify cell viability. Its safety enables reuse of the same cell population, making it particularly valuable in tissue engineering and biomaterials research. Although typically used as an endpoint assay, CCK-8 can also be applied repeatedly within the same experiment to monitor temporal changes in cellular activity.

Historical Context: From MTT to CCK-8

The development of cell viability assay began with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, introduced by Tim Mosmann in 1983. As one of the earliest and most influential methods, it established the foundation for modern colorimetric assays that rely on the conversion of a colorless substrate into a colored product by viable cells, not by dead cells or the culture medium. Tetrazolium salts are particularly suited for this purpose, as they are reduced exclusively by active mitochondrial enzymes in living cells.

The MTT assay is a rapid colorimetric technique that quantifies viable cells by measuring mitochondrial activity. Specifically, active mitochondria reduce the yellow MTT salt to an insoluble purple formazan product, the intensity of which is directly proportional to the number of living cells and can be measured via spectrophotometry.

Despite its widespread use, the MTT assay has some limitations:

• The formazan product is water-insoluble and requires solubilization using solvents like dimethyl sulfoxide (DMSO).
• It exhibits cytotoxicity, limiting downstream applications.
• The process is labor-intensive due to additional dissolution steps.
• It may interfere with components of the cell culture medium.

To overcome these limitations, researchers developed modified tetrazolium salts such as WST-1 and WST-8, which produce water-soluble formazan. WST-8, used in the CCK-8 assay, is reduced by cellular dehydrogenases to form a soluble dye, allowing for real-time metabolic monitoring with minimal cytotoxicity. Among tetrazolium-based assays, WST-8 has shown superior sensitivity in detecting cell viability.

Principle And Advantages Of The CCK-8 Assay

The CCK-8 assay is a chromogenic method used to evaluate cell viability by measuring the reduction of yellow WST-8 to orange formazan in metabolically active cells, with the color intensity correlating to the number of viable cells. The assay relies on detecting NAD(P)H-dependent dehydrogenase activity by the action of an electron carrier called 1-methoxy-5-methylphenazine dimethyl sulfate. This process makes it a sensitive indicator of cellular metabolic status, which is closely tied to cell function and phenotype.

Key advantages of the CCK-8 assay include:

• High sensitivity and accuracy: The CCK-8 assay offers superior sensitivity and precision compared to traditional tetrazolium-based assays like MTT. This is largely due to the WST-8 substrate’s high molar extinction coefficient, which enables the detection of low cell numbers with greater accuracy. For instance, WST-8 can detect as little as 0.3 nmol of NAD(P)H, making it approximately five times more sensitive than conventional UV-spectrophotometry. Unlike MTT, the formazan product of WST-8 is water-soluble, eliminating signal interference caused by precipitate formation and thereby improving assay reliability.

• Simplified procedure: The CCK-8 assay involves a straightforward, no-wash protocol without the need for solubilization steps. After a short incubation period (typically 1–4 hours), absorbance can be measured directly. This simplicity makes it ideal for high-throughput applications and reduces hands-on time.

• Low cytotoxicity: WST-8-based assays are non-toxic and non-radioactive, allowing repeated measurements and long-term studies on the same cell population. For example, in a study assessing the proliferation of rat mesenchymal stem cells in 2D and 3D collagen hydrogels over four weeks, the non-toxic nature of the WST-8 reagent enabled continuous monitoring without compromising cell viability. This feature is particularly advantageous for time-course experiments and follow-up assays in regenerative medicine and tissue engineering.

Applications In Biomedical Research

A cell viability assay measures cellular metabolism and enzyme activity to determine cell health, making it useful for evaluating drug toxicity and screening potential treatments. The CCK-8 assay has evolved in various fields of biomedical research, such as:​

• Drug screening: Its high sensitivity and compatibility with high-throughput formats make it ideal for evaluating the cytotoxic effects of potential therapeutic compounds. WST-8 reduction is highly dependent on cellular glucose metabolism. For example, a study showed that when glucose was withdrawn, it significantly inhibited dye reduction, which could be reversed by glucose re-addition. This reduction defect was not due to cell death, as confirmed by ATP assays. This highlights the assay’s potential for identifying drugs that target cellular glucose metabolism. ​

• Cancer research: The CCK-8 assay is widely employed to evaluate the efficacy of anticancer agents by quantifying their effects on tumor cell viability. In one study, the combined impact of curcumin and cisplatin (DDP) on HT29 colorectal cancer cells was investigated. Results from the CCK-8 assay revealed that the combination treatment significantly decreased cell viability compared to either compound alone. This reduction was dose-dependent, and subsequent analyses showed an increase in apoptosis within the combination group, indicating that curcumin may enhance the anticancer activity of cisplatin.

• Toxicology studies: The WST-8 assay is a valuable tool for evaluating the cytotoxicity of environmental toxins and nanoparticles, aiding in safety assessments. One study examined the acute effects of titanium dioxide nanoparticles (TiO₂NPs) on human breast adenocarcinoma (MCF-7) cells. Results showed a concentration- and time-dependent decline in cell viability following exposure to TiO₂NPs at 25 and 50 µg/mL for 24 and 48 hours. These findings highlight the assay’s sensitivity in detecting subtle viability changes caused by nanoparticle exposure.

Conclusion

The evolution from MTT-based assays to the advanced WST-8-based CCK-8 assay highlights a significant leap in improving sensitivity, reducing cytotoxicity, and simplifying cell viability testing. With its non-toxic nature, simple protocol, and high-throughput compatibility, the CCK-8 assay has become a vital tool for accurate, real-time assessment of cell proliferation and viability. Its wide applicability from cancer drug screening to stem cell and toxicology studies makes it indispensable for modern biomedical research.