Cell-Based Assays In Screening Of A Variety Of Drug Compounds

Introduction:

Cell-based assays are assays that are used commonly for the screening of a variety of drug compounds in order to determine if the compounds have any effect on cell proliferation or if they display cytotoxic effects (Riss et al., 2013). In addition, cell-based assays can also be used to measure target receptor binding and signal transduction effects that involve trafficking of cellular components, the expression of genetic reporters or monitoring organelle functions (Riss et al., 2013). In order to determine the effects in cell proliferation or any cellular cytotoxicity, this can be done through the measurement of viable cells present at the end of the experiment compared to the number of viable cells present at the beginning of the experiment. There are copious amounts of different cell-based assays such as tetrazolium reduction assays, protease marker assays, resazurin reduction assays and ATP detection assays (Riss et al., 2013). Different tetrazolium reduction assays are available namely, MTS cell proliferation assay and MTT assay which uses compounds MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) respectively (Riss et al., 2013).

MTS cell proliferation assay is the most commonly used tetrazolium reduction assay and is a colorimetric method for the quantification of viable cells, which is based on the reduction of the MTS tetrazolium compound by viable cells to generate a colored formazan product that is soluble in cell culture media (Berridge et al., 2005). This conversion is done by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells (Berridge et al., 2005). MTS cell proliferation assays are performed by the addition of MTS reagent into the cell culture media and require no washing or solubilization step (Riss et al., 2013). There are many different cell lines that can be studied using MTS cell proliferation assays, namely, HEK 293T cells, LNCaP prostate cancer cells and MCF-7 breast cancer cells. HEK 293T cells are human embryonic kidney cells that are highly transfectable and contain a SV40 T-antigen (Lin et al., 2014). In addition, MTS cell proliferation assays are used to understand cell proliferation in response to external stimulus i.e. cytotoxic reagents, cytokines, and growth factors (Berridge et al., 2005). Thus, this assay can be used to analyse cytotoxic compounds such as anticancer drugs, pharmaceutical compounds and mAbs.

Alternatively, to MTS cell proliferation assays, the MTT assays can be used and it works in that NAD(P)H-dependent cellular oxidoreductase enzymes that are in viable cells can reduce the tetrazolium dye MTT, to its insoluble formazan, which has a purple colour (Riss et al., 2013). Under defined conditions, it can reflect the number of viable cells present and can be used to measure cytotoxicity or cytostatic activity of potential medicinal agents and toxic materials. This method is less expensive than MTS cell proliferation assay, however, MTS is more efficient than MTT and produces water-soluble formazan that does not require DMSO dissolution and it is faster in that 2-3 hours are needed for MTS reactions where as MTT assays require 4 hours (Riss et al., 2013).

The mouse-derived IgG monoclonal antibody (mAb) used in this experiment was previously purified and characterised by affinity chromatography and high-performance liquid chromatography. Following purification and characterization, an enzyme-linked immunosorbent assay and Biacore surface-plasmon resonance was used to test the functionality of the mAb in vitro and to show the IgG mAb had moderate affinity for its IgG-antigen target. This investigation aimed to determine the cytotoxic effects of the previously purified biopharmaceutical IgG mAb (test) compared to two competitive antibodies (1 and 2) on a human cell line (HEK 293T) using a MTS cell proliferation assay. Two 96-well cell culture plates were produced in that for each cell plate, 100 L of HEK 293T cells were mixed with 100 mL of Tryptan blue and a Countess II FL cell counter was used to count the number of cells and the cell viability. Originally 400 mL of Tryptophan blue was supposed to be used instead of 100 mL in order to count the cells manually using a haemocytometer grid, however, due to time restraints, the volume was reduced, and the Countess II FL cell counter was used. The HEK 293T cells were seeded into 96-well cell culture plates and the test, competitor 1 and competitor 2 were added to the cells at concentrations 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2, 1, 0.5 g/mL and were left to incubate overnight at 37oC with 5% CO2. MTS reagent was added to the wells and incubated for 1 hour instead of 45 minutes as stated in protocol. Following incubation, a stop solution was added to the wells and the absorbances were measured at 492 nm. Percent cell viability was calculated. The data produced was analysed using Microsoft Excel and IBM SPSS Statistics 22. One-way ANOVAs were used to measure any significant differences. Significant differences are observed when p

Results:

The absorbances measured from the two 96-well cell culture plates allowed for the comparison of cell viability at different concentrations of the buffer, test IgG, competitor 1 and competitor 2. The average absorbances of both 96-well cell culture plates are represented in Table 1, which in turn allowed for the generation of a log-dose vs response (cell viability) graph as shown in Figure 1.

The untreated control cells and no cell control had an absorbance of 0.862 and 0.500 respectively. The cell viability remains constant at ±100% at all concentrations when the buffer is used, however, at concentration 500 g/mL the cell viability is ±110%, which could be due to a higher cell density used compared to the test IgG, competitor 1 and competitor 2. A similar trend is produced with the test IgG, competitor 1 and competitor 2 in that the cell viability decreases as the concentration increases. The cell viability remains constant for the test IgG, competitor 1 and competitor 2, however, at concentrations 125 mg/mL and higher the cell viability decreases. At concentration 500 mg/mL, the largest decrease in cell viability is observed with competitor 2 having a cell viability of ±30%, followed by competitor 1 with a cell viability of ±65% and the test IgG with a cell viability of ±70%. Significant differences were observed at concentration 500 mg/mL between the buffer and competitor 2 (p=0.016) and at concentration 15.6 mg/mL between the test IgG and competitor 2 (p=0.012). (Key: buffer – , test IgG – , competitor 1 – and competitor 2 – ). The log-dose vs response (cell viability) curve represents a similar trend between the test IgG, competitor 1 and competitor 2 in that as the concentrations increase, the cell viability decreases. A constant cell viability of ±100% is represented by the buffer. A significant difference was observed at concentrations 500 g/mL between the buffer and competitor 2 (p=0.016) and significant difference was observed at concentrations 15.6 g/mL between the buffer and competitor 2 (p=0.0012). The largest decrease in cell viability was observed at concentration 500 g/mL resulting in a cell viability for the test IgG, competitor 1 and competitor 2 being ±30%, ±65% and ±70% respectively.

Table 1: The averages of two 96-well cell culture plates absorbances at different concentrations of the buffer, test IgG, competitor 1 and competitor 2.

Reagents Concentrations of reagents used (mg/mL)

• 500 250 125 62.5 31.3 15.6 7.8 3.9 2.0 1.0 0.5
• Buffer 0.827 1.098 0.808 1.001 0.605 0.835 0.999 0.933 0.939 0.777 0.837
• Test IgG 0.683 0.989 0.875 0.895 0.576 0.879 0.982 0.947 0.814 0.857 0.741
• Competitor 1 0.659 0.945 0.906 0.969 0.664 0.815 0.990 0.923 0.911 0.846 0.674
• Competitor 2 0.532 0.960 0.688 0.854 0.626 0.709 0.963 0.948 0.898 0.846 0.642

Discussion:

Cell-based assays like that of the MTS cell proliferation assays are simple, convenient and high-throughput and enable to determination of cell proliferation and cellular cytotoxicity. The aim of this investigation was to determine if there were any cytotoxic effects of the test IgG compared to competitor 1 and competitor 2 on a HEK 293T cell line using a MTS cell proliferation assay. From the results obtained the buffer has shown to have constant cell viability at ± 100% at the changing concentrations, however, at the highest concentration, 500 g/mL, a higher cell viability was observed. This may be a result of a higher cell number present in the wells at the start of the investigation. An increase in cell number may have been caused by pipetting errors. However, if the difference in cell number was noted for the buffer compared to the test IgG, competitor 1 and competitor 2, more MTS reagent would of be required in the wells with buffer in order to ensure similar cellular metabolism reactions. The test IgG, competitor 1 and competitor 2 all produced a similar trend in that as the concentrations increased, the cell viability decreased. At the highest concentration, 500 g/mL, competitor 2 produced the largest cell viability decrease, resulting in only ±30% cell viability remaining. This was followed by competitor 1 with a decrease in cell viability to ±65% and then the test IgG with remaining cell viability of ±70%. This therefore shows that at higher concentrations, competitor 2 has the highest cytotoxicity, compared to the test IgG being the least toxic. Significant differences were observed at concentration 500 g/mL between the buffer and competitor 2 (p=0.016) and at concentration 15.6 g/mL between the test IgG and competitor 2 (p=0.012). At the highest concentration of buffer, no cell death should be observed as the buffer has no toxic effects. By treating the HEK 293T cells with competitor 2, the cells did not have 100% cell viability at the lowest concentration compared to the buffer, test IgG and competitor 1. These differences may be a result of differences in cell numbers at the start of the investigation.

From the ideal results, significant differences were also found at 500 g/mL between the buffer and the test IgG (p=0.015). A similar trend was also observed in that as the concentration increases, the cell viability decreases. At the highest concentration, competitor 1 was the most toxic, followed by competitor 2 and lastly test IgG. This is different to the results obtained in this investigation. There is similarity between the investigation results and the ideal results as the test IgG is the least toxic compared to both competitor 1 and competitor 2. However, as the test IgG has little effect on decreasing cell viability at high concentrations, this could mean that the test IgG does not have an effect on cell proliferation. Therefore, if the test IgG was to be used therapeutically, it would not be efficacious in the target disease because of the lacking effect on cell viability. The investigation results and ideal results however do not reflect similarity when competitor 1 and competitor 2 are compared. In this investigation, competitor 2 was found to be the most effective at reducing the number of viable cells, however, the ideal results found competitor 1 to be the most effective at reducing the number of viable cells.

This investigation can be improved by ensuring pipetting errors do not occur when producing the serial dilution and when seeding the HEK 293T cells into the 96-well cell culture plates. It is also important to ensure that at the start of the investigation, the HEK 293T cell sample used contains more than 95% viable cells as during this investigation the HEK 293T cell sample only contained 66% viable cells.

In conclusion, the previously purified test IgG was found to have little toxic effects on the HEK 293T cell line and competitor 2 was found to be the most toxic. However, if test IgG was to be used therapeutically, concentrations higher than 500 g/mL will be required. Furthermore, due to the differences between the ideal results and this investigation regarding if competitor 1 or competitor 2 is the most toxic, the investigation should be repeated. Alternative methods like that of MTT tetrazolium reduction assays or ATP detection assays may be useful.