Utilization of cell-based assays

The time and costs of drug discovery have increased over the past twenty years. Thus, it is of paramount importance that the selection and concomitant dismissal of compounds is achieved earlier in the drug discovery process. The utilization of cell-based assays in primary screening, secondary screening and ADMET (adsorption, distribution, metabolicity, excretion and toxicity) facilitates the selection of compounds that have favourable biological properties. Cell-based assays are primarily used to identify chemical entities that elicit a specific biological response such as the inhibition of a cell-surface receptor. Moreover, cell-based assays are used to discern compounds that have non-desirable biological effects; pro-apoptotic and/or cytotoxic compounds are eliminated from the screening process. The importance of elucidating toxic compounds earlier in the drug discovery process is highlighted by the fact that toxicity accounts for the elimination of a third of compounds at the ADMET stage. Due to the vast cost involved in progressing a compound to the ADMET stage it is essential that toxicity/apoptosis screening is performed as early as possible in the drug discovery process. As a result, the incidence of cell-based assays utilized in primary/secondary screening has more than trebled in the past decade.  

The advantages of cell-based assays are:

1. biological relevance e.g. for intracellular targets the compound must first cross the cell membrane
2. cell-based assays can be performed rapidly with large data output
3. time and cost saving e.g. cell lines that endogenously express proteins of interest circumvent expense of generating a cell line expressing the desired protein

The focus of cell-based assays is moving toward high-content screening (HCS) and high-content analysis (HCA). For each compound, a specific biological response can be assessed simultaneously with the cytotoxic/pro-apoptotic propensity of that compound. There therefore exists the need for not only a high resolution, multi-wavelength imaging system, but also an assay management system to streamline compound screening. Genetix has developed the CellReporter to rapidly scan live cells in micro-well plates using a bright-field imaging system and at up to 6 different fluorescent wavelengths; a 384-well plate can be scanned in bright-field and at 2 fluorescent wavelengths in under 10 minutes. In synergy with the imager Genetix continues to develop a suite of cell-based assays to utilize the full potential of this new system.

Cytotoxicity

It is vitally important that the toxicity of compounds is determined early in the screening process so that time and money are saved by eliminating cytotoxic compounds. One of the Genetix Cytotoxicity assays utilizes a TxRED-detectable dead cell dye; the assay also stains the nuclei of all cells with a DAPI-detectable dye. To model the effects of a cytotoxic compound, adherent cells were incubated with the DAPI-detectable dye, the TxRED-detectable dye and ethanol.

A                                                              

C

D

Figure 1. Live cell imaging to ascertain cytotoxicity.
Adherent CHO were plated and proliferated to approximately 80% confluence. To mimic the effects of a cytotoxic compound, cells were incubated with a titration of ethanol. The TxRED-detectable dye stains the nucleus of all dead cells (DAPI-detectable dye images not shown). (A) Untreated cells incubated with dye. (B) Cells were incubated with 10% ethanol for 10min prior to the addition of dye. (C) Cells were incubated with 20% ethanol for 10min prior to the addition of dye. Ethanol titration mimics the differing propensity of cytotoxic compounds and can be analyzed by assessment of dead cell nuclear staining.

Software Analysis

We demonstrate the high resolution (0.8mm at 10X objective) quality of images produced on the CellReporter and examples of some of the assays currently under development. The bright-field imaging capacity facilitates the verification of cell confluence and cellular morphology pre- and post-assay. Plates can be imaged at up to 6 different wavelengths at high speed.  Software algorithms and an assay management suite will offer the user complete control throughout cell-based assay projects. Genetix CellReporter can be utilized with the following applications: