In the hunt for new drugs it is particularly important to understand if your compounds could have a negative impact on cellular health, even if they do interact with your target of interest. This requirement is determined with a variety of different measures and steps within the drug discovery process. One early and quick method is the use of in vitro cellular viability assays.
A group from the University of Otago compared some of the different in vitro cellular viability assays available in the following recent publication (Single, A. et al J Biomol Screen 2015, in press).
The authors wanted to investigate any differences between the more conventional viability endpoint measurements, such as resazurin based (a fluorescent based measurement) and CellTiter glo® (a luminescent based assay) with nuclear counting techniques (fluorescence based imaging technique). Further comparisons were made with the more recent developments from xCELLigence (an electrode impedance measurement) and IncuCyte (live cell imaging) assays. Both these latter methods can measure in kinetic intervals, which may offer a different insight by measuring cell growth rates in response to treatment with different compounds.
All the assay methods were tested using the MCF10A cell line and additionally the CDH1-negative isogenic line for which the compound vorinostat would be synthetically lethal. Taxol was used as a non specific toxic compound control.
First the endpoint assays were compared, the nuclear counting method showed a greater detection of reduced viability compared to the resazurin and CellTiter glo® methods. Another point of interest was that the CellTiter glo® showed the lowest sensitivity of all three measures. This was quite surprising given my experience of the use of this assay format and as it is a widely quoted assay in published literature. It would be interesting to see if the same trend occurs in other cell lines or if it was specific to this cell type. Also, a wider panel of compounds would be a useful adjunct to this work.
The Kinetic based platforms (the IncuCyte and xCELLigence) were used to determine proliferation rate during the logarithmic growth phase of the cell lines and also at full confluence of the cells. During the log phase, both systems reported reduced growth rate matching the different dose additions of the compounds. For the xCELLigence system slower rates were achieved, but the authors suggested this could be due to lower cellular adhesion of this cell type and thereby reducing the number of cells being detected. Once the cells had reached full confluence, the difference between treatment and non treatment of compound became very small, so it appears the use of these kinetic based platforms requires measurements in the log phase to generate the most reliable data.
As the group wanted to develop a method to identify synthetically lethal compounds against this cell line, they further investigated the use of a multiplexed assay format to overcome the limitations of the different methods they encountered. They combined both the resazurin and nuclei counting methods with the IncuCyte measurement and determined viability ratios with the results.
Using the multiplexed format the authors could determine the synthetically lethal effect of the vorinostat at both the log phase and full confluency of the cells. It was also noted that certain Taxol concentrations produced reduced viabilities during the growth phase of cells, but when full confluence endpoint was measured the cells had recovered to generate the similar readings as DMSO controls. This again highlights the advantage of using a kinetic measurement to discern these very subtle differences.
In summary the authors suggested the use of the multiplexed assay format as the most sensitive way to determine synthetically lethal compounds, but if that was not possible, they suggest the use of nuclear counting as it appeared to generate the most sensitive endpoint result. Overall, this was an interesting publication, which has challenged some of my preconceptions about the most optimal assay to use for this type of experiment. It is also interesting to see these more recently developed methods in real world action.
Blog written by Gareth Williams