Assays that quantitate the effect of small molecules on cellular behavior are powerful tools in drug discovery. The ability to assess compounds in a physiologically relevant environment is particularly informative, as the assays simultaneously evaluate compound permeability, toxicity, and potency. Many cell-based assays report on global cellular activity rather than focusing on a specific target, increasing the chances for identifying compounds that elicit a desired cellular response.

One of the most prevalent cell-based assays in drug discovery aims to understand compound-driven toxicity, which is a main contributor to the high failure rate of drug candidates. Toxicity is often measured by assessing the number of live cells (a “viability” assay) or measuring cell death (a “cytotoxicity” assay). Notably, these assay methodologies largely require certain reagents to report on viability or cytotoxicity, and these reagents intrinsically impact cellular health, complicating data analysis. Choosing the right assay methodology for your viability or cytotoxicity assay can be a life-or-death decision for your cells and project.

To guide this critical decision, here are three important aspects to consider when selecting a methodology for your next viability or cytotoxicity assay.

Establishing a relevant cellular model

Viability and cytotoxicity assays begin with the selection and production of an appropriate cell line that adequately models the target disease and will provide a readout that will inform on the impact of the compound on cellular health. Different cell types, including primary cells, immortalized cells, human cells derived from stem cells, and engineered cell lines (e.g., addition of a reporter gene or a live cell imaging tag), require unique considerations based on cellular behavior, maintenance, and handling. It is also important to consider the types of cells that the potential drug will encounter to understand whether any toxicity is unique to a specific cell type or general, impacting nearly all cells. Many cytotoxicity assays report on a particular cell death pathway such as apoptosis or necrosis. Ensuring that the pathway of interest is induced in the cell line to be studied will therefore be important. Alternatively, if the project aims to understand how a compound slows proliferation rates, which is often the case for new oncology drugs, understanding the compound half-life and doubling time of the cell line is important to establish a protocol to properly evaluate the compounds. Moreover, subtle factors such as cell culture media, volume, gas exchange, and liquid evaporation can influence cellular behavior and the impact of small molecules. Ensuring a consistent and robust platform to account for these subtilties will be valuable for generating robust, reproducible and relevant data.

Figure 1: Cell health – Available assays and their proxy measure of viability/cytotoxicity

Selecting a cell health proxy to measure

Cellular health can be measured through several pathways (Figure 1). Healthy viable cells contain abundant metabolic indicators including ATP, glutathione, and active NAD(P)H-dependent enzymes. In response to stress such as the presence of a small molecule, the cell may decrease the activity of these factors and increase the activity of other enzymes, such as caspases, or present other phenotypes such as chromosome condensation and membrane blebbing. Upon cell death, the membrane is compromised, releasing cytoplasmic enzymes and other cellular debris. Choosing which marker of cellular health is most relevant will depend on the test compound behavior and its cellular targets, the chosen cell line, and the capabilities of the lab to measure these markers. In many cases, outsourcing these assays to contract research organizations (CROs) that have an established infrastructure and experts in cellular assay development can aid in accelerating the process and ensuring quality data for your compounds.

Critical considerations for assay development

It is important to note that for each cell health proxy measure, there are extensive options for commercially available kits that follow the same principle but may significantly differ in mechanism, readout (absorbance vs. fluorescence), and stability. Each of these factors can play a role in the quality of the data generated and how the results should be interpreted. Moreover, the reagents themselves may exhibit inherent toxicity, and distinguishing test compound toxicity relative to the toxicity of the reagents used to measure cell health can be challenging. It is therefore important to understand the specific goals of the assay to guide the reagent choice. As an example, tetrazolium salts and resazurin are two available families for measuring NAD(P)H-dependent redox activity in a cell with several derivatives available for each family. Since these compounds intrinsically impact cellular health differently, it can be valuable to test multiple reagent kits to determine which generates the most robust data for your project. Understanding assay reagents at this level simplifies the decision-making process for measuring cell health and adds confidence in the generated data, accelerating drug discovery programs and taking another step towards the clinic.