Grigorenko Svetlana Evgenyevna

PhD in Pedagogy, Associate Professor of the Department of Foreign Languages and Professional Communication,

Belgorod State National Research University, Belgorod, Russia

E-mail: [e-mail]

The article analyses drug screening as an important step in drug design which allows determining biological activity of a drug in vivo and in vitro. It can be performed by means of different biochemical methods, and the author emphasizes one of them, which is the MTT assay. It is presented as a colorimetric assay based on measurement of cell metabolic activity. The article attempts to contribute to the debates upon application of the method, as well as intracellular mechanisms of the assay. Besides, the advantages and the disadvantages of the examined method are highlighted in the article.

Keywords: сell culture, сell viability, сolorimetric assay, drug screening, MTT assay.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is one of the most common methods for measuring cell viability, radiosensitivity and cytotoxicity of compounds in studies in vitro [Stockert et al. 2018]. These parameters are used while screening chemical agents for inhibitory effect towards different types of cells and determining antineoplastic drugs and their doses for chemotherapeutic purposes [Tominaga et al. 1999].

The MTT reagent is a tetrazolium salt, reduced by enzymes of metabolically active cell to water-insoluble purple formazan. After crystal formation, the culture medium is replaced with an organic solvent and extraction takes place with a measurement of the solution optical density following after. The procedure described by Mosmann in 1983 suggests that the amount of the formed formazan is proportional to the number of viable cells with functioning mitochondria. Thus, high purple color intensity indicates a high rate of cell survival, whereas low intensity reflects a decrease in cell number and therefore cytotoxicity of tested compounds [Mosmann 1983].

The first hypothesizes, explaining the mechanism of enzymatic reduction of the tetrazolium salt, associated its transformation with localization in mitochondria and mitochondrial dehydrogenase activity. Thereby the MTT assay was considered as a method of mitochondrial activity determination. Slater et al. contributed to the establishment of the theory by studying interaction between tetrazolium salts and the respiratory chain. They showed that the reduction of MTT is carried out by enzymes of the inner mitochondrial membrane using succinate as an electron
donor [Stockert 2018].

However, according to more recent researches, only part of the final product is associated with mitochondria, and most of the formazan accumulation is observed in other organoids, cytoplasm and in a cell membrane. For instance, formazan granules were found in lipid droplets, which can be an evidence of them being the place of formazan final intracellular localization [Berridge, Tan 1993].

Endosomes/lysosomes also were considered as the place of MTT-formazan accumulation, but the hypothesis hasn’t been verified by data derived from studying cell ultrastructure during the process of formazan formation and from the fluorescent analysis [Stockert 2018].

Currently, the enzymatic reduction of MTT-reagent is viewed as a result of NADH (nicotinamide adenine dinucleotide (reduced form)) (and NAD(P)H- (nicotinamide adenine dinucleotide phosphate (reduced form)) dependent oxidoreductases activity in the endoplasmic reticulum (ER), but the exact mechanism of formazan granules formation remains unknown[Berridge,
Tan 1993].

Like any other method, MTT-assay has its own advantages and also application restrictions concerning both reagent properties and protocol steps.

Currently, the MTT assay is known to be a simple and safe method for determining drug cytotoxicity and cell viability with high accuracy comparing to the dye exclusion test that has been used before [Riss et al. 2016].

MTT assay can be performed using stable cell lines as well as primary cell cultures [Van Meerloo et al. 2011].

The potential application of MTT-assay in a drug screening can lie in determining the following characteristics:

1) cytotoxic/cytostatic effect of a drug on different cell lines;

2) in vitro drug sensitivity;

3) in vitro drug resistance [Bahuguna 2017; Morgan 1998].

The main disadvantage of the MTT assay is the necessity to consider many factors while using the method for purposes of a specific experiment and some data inaccuracies due to it. For instance, prolonged incubation with the MTT-reagent allows to increase the final optical density of the solution and achieve better sensitivity but in the same time, the total incubation time is limited by the cytotoxic effect of the reagent using the cell energy reserves for a signal generation. Furthermore, exocytosis of formazan crystals leads to the cell
structure and activity disruption, causing an inability to use these cells in another
assay [Riss et al. 2016].

Compared to other colorimetric methods based on water-soluble tetrazolium salts (MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), WST(water-soluble tetrazolium salts), the MTT assay requires more steps since the final product, formazan, has to be
extracted with organic solvents such as DMSO(dimethyl sulfoxide)/isopropanol/etc. [Van Meerloo et al. 2011].

It is also important to consider the impact of medium components on formazan crystal formation. Transformation of the tetrazolium salt might be carried out not only by cell enzymes but also by antioxidants and vitamins present in a culture medium. Also, it’s been shown that serum albumin, which addition is necessary for the growth of some cell lines, can cause obtaining false-positive results since its free cysteine residue is capable of reducing the
MTT reagent [Tominaga et al. 1999].

The other parameter that needs to be controlled is the pH level of the medium when inserting MTT reagent into wells and extracting formazan with a solvent – the presence of acidic residues in the formazan /DMSO system leads to obtaining lowered absorbance values [Mosmann 1983].

Metabolically inactive cells produce formazan in undetectable quantitates, so the test objects need to be chosen thoughtfully according to their physiological characteristics [Morgan 1998].

While testing for cytotoxicity, the researches have to consider the fact that the substance might affect the formazan formation, e.g. by increasing non-specific MTT reduction or directly interacting with a tetrazolium salt. For that reason, some cases of MTT assay use require additional verification with other assays (CCK-8 (cell counting kit-8), ATP (adenosine triphosphate), etc.), and obtained results need to be interpreted with a certain level of caution [Riss et al. 2016].

Thus, MTT assay has been considered a golden standard for cell viability assays for a long time, being one of the first colorimetric methods allowing to measure metabolic activity rate of different types of cells, normal or malignant. This assay can serve as a useful tool in high-throughput drug screening and allows obtaining reliable data but has its own limitations and appliance restrictions that are important to know when choosing this method for specific research purposes.

 

References

1. Bahuguna, A. (2017). MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh Journal of Pharmacology, 12. – Pp. 115-118.

2. Berridge, M.V., Tan, A.S. (1993). Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Archives of Biochemistry and Biophysics, 303/2. – Pp. 474-482.

3. Morgan, D.M. (1998). Tetrazolium (MTT) Assay for Cellular Viability and Activity. Methods in Molecular Biology, 79. – Pp. 179-183.

4. Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. Journal of lmmunological Methods, 65. – Pp. 55-63.

5. Riss, T.L. et al. (2016). Cell Viability Assays. Assay Guidance Manual. – [online]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK144065. (Accessed: 13.01.2021)

6. Stockert, J.C. (2018). Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochemica, 120/3. – Pp. 159-167.

7. Tominaga, H. et al. (1999). A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Analytical Communications, 36. – Pp. 47-50.

8. Van Meerloo, J., Kaspers, G.J., Cloos, J. (2011). Cell Sensitivity Assays: The MTT Assay. Cancer Cell Culture: Methods and Protocols, Second Edition, Methods in Molecular Biology, 731. – Pp. 237-245.