2.2.10 Cytotoxicity assay

The critical factor that allows any compound to proceed successfully during drug discovery is selective toxicity to pathogens and non-toxicity to human cells. The assay was performed under the standards set by International Organization for Standardization (ISO) and Economic Cooperation and Development (OECD), to ensure accurate and reproducible results. The cytotoxicity of the lead compound 2d was evaluated against HEK-293. Before evaluating the cytotoxicity of the compound, the cells were grown to reach a confluency of 70%–80%. The culture techniques used were in accordance with the cell culture technique guidelines regulated by the International Cell Line Authentication Committee (ICLAC), American Type Culture Collection (ATCC), and the European Collection of Authenticated Cell Cultures (ECACC).

Human Embryonic Kidney Cell lines, HEK-293 were purchased from National Centre for Cell Sciences (NCCS), Pune, India. The cell lines were cultured in DMEM (supplemented with 10% foetal bovine serum) in a Carbon Dioxide incubator with 98% humidity and 5% CO₂ at 37°C.

Thawing of frozen cells: HEK-293 cells stored at −80°C were thawed by removing the frozen cryovial and thawing it in 37°C water for 1–2 min until no visible was seen in the vial. The vials were swirled gently to ensure that the cells are fully thawed. Once the cells were thawed, the vial was removed from the water bath and wiped with 70% ethanol to sterilize the outside of the vial.

Cell Culture and Expansion: The thawed HEK-293 cells were then transferred to a sterile hood and the contents of the vial were transferred into a sterile tube containing 9 mL of pre-warmed growth medium Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, and 1% penicillin-streptomycin. This was followed by centrifuging the tube at 1,000 rpm for 5 min to pellet the cells. The supernatant was aspirated and the cell pellet and resuspended in 2 mL of fresh pre-warmed growth medium. The cell suspension was transferred to a T75 culture flask and incubated at 37°C with 5% CO₂ until they reached the desired confluency of 70%–80%.

Harvesting of cells: Once the cells had reached the desired confluency, they were harvested using standard trypsinization techniques. A small volume of Trypsin-EDTA solution was added to the culture flask and incubated at 37°C for 2 min. During incubation, the Trypsin cleaves the proteins that attach the cells to the culture surface, causing the cells to detach from the surface. The culture medium was removed from the culture dish, and the cells were washed with phosphate-buffered saline (PBS) to remove any residual serum. After incubation, an equal volume of culture medium containing FBS and DMEM was added to neutralize the Trypsin and stop the enzymatic activity. The cells were then collected by gently tapping or swirling the culture dish and transferred to a centrifuge tube. The cells were again pelleted by centrifugation, and the supernatant is discarded. The cells were resuspended in a fresh culture medium, counted, and seeded into a new culture flask. The harvested cells were then counted using a hemocytometer. A predetermined number of cells were seeded into 96-well optimized for the cytotoxicity assay being performed.

The method was carried out as described earlier (Adan et al., 2016). The 100 µL of HEK-293 cells were seeded into the 96-well microtiter plate and allowed to grow for 24 h at 37°C with 5% CO₂. The cells were treated with 6 concentrations of the 2d compound 4, 8, 16, 32, 64, and 128 μg/mL dissolved in DMSO for time points 24, 48, and 72 h to determine the time and dose dependent effect of the compound on cell viability. Untreated control wells received only DMSO. After the given time points, the number of viable cells was determined. Briefly, the tissue culture medium was removed from the 96-well plate and replaced with 100 µL of fresh medium then 20 µL of the MTT stock solution (5 mg of MTT in 1 mL of PBS) was added to each well including the untreated controls. The 96-well plates were then incubated at 37°C and 5% CO₂ for 4 h. An aliquot (85 µL) of the medium was removed from the wells, and 150 µL of dimethyl sulfoxide was added to each well to dissolve the insoluble formazan crystals formed by viable cells and mixed thoroughly with the pipette and incubated at 37°C for 10 min. The optical density was measured at 590 nm with a microplate reader to determine the number of viable cells based on the selective ability of viable cells to reduce the tetrazolium component of MTT into purple-colored formazan crystals. The colored formazan intensity was measured at 570 nm absorbance in the micro-plate reader and growth percentage inhibition was calculated using the formula:

The results expressed were the average values of three experiments (±SD). Reduction of MTT to formazan crystals is mediated by the mitochondrial enzyme succinate dehydrogenase of cells the viable cells. The color of the formazan crystals that form after the reduction of MTT by viable cells indicates the level of cell viability and, hence, the cytotoxicity of the test compound. The formazan crystals appear as purple or dark blue precipitates in viable cells. The intensity of the color is directly proportional to the number of viable cells in the culture. Conversely, a decrease in the intensity of the color indicates a decrease in the number of viable cells and, hence, an increase in cytotoxicity. A complete absence of color indicates that all the cells in the culture have died, and the test compound is highly cytotoxic to the cells.