Despite a growing awareness of the value of three-dimensional (3-D) cell culture models for drug discovery, cell-based assays for high-throughput screening (HTS) are typically limited to monolayers of adherent cells. 3-D cell culture models are clearly important for understanding the processes involved in converting a noninvasive phenotype into an invasive one and for evaluating therapeutic agents.
Although there is considerable evidence demonstrating that the extracellular matrix and tissue architecture play an important role in the normal functioning of cells and in tumorigenesis, the formation of 3-D colonies in semi-solid media presents many challenges associated with performing and imaging this assay in a form that meets HTS requirements.
3-D cell culture models are especially challenging for microscope-based systems because of the need to tile multiple regions to get sufficient field-of-view and the need to stack multiple z-heights to adequately sample thick media. Moreover, fast and robust computational tools for image analysis of such samples are lacking for the HTS environment.
At Blueshift Biotechnologies (www.blueshiftbiotech.com), a solution to these problems has been achieved with an optimized assay system that utilizes the IsoCyte™ scanning cytometer. The colony assay is set up in 96-well, black-walled plates with square wells and a glass bottom (MatriCal(www.matrical.com)) that results in better control of the media layers as well as improved optical access and image quality.
A simple one-step colony-staining protocol has been developed using LavaCell™ (Active Motif(www.activemotif.com)). LavaCell stains cellular membranes of live or dead cells by reacting with free amine groups on proteins. It produces a bright fluorescent signal upon 488-nm laser excitation with a peak emission at 610 nm. The IsoCyte is then used to scan over full plates, analyzing whole-well areas in a single pass in approximately five minutes.
The IsoCyte has a proprietary optical system with a large depth of field (400 µm) and binocular setup making it well-suited for 3-D colony measurements for HTS and drug discovery efforts (Figure 1). The system can image cell colonies at several micron resolution over whole plates without need of focus adjustment or acquisition of multiple z-heights.