Expert view: Three-dimensional cell cultures as predictive tools in early drug discovery

Three-dimensional cell cultures (spheroids, organoids) are becoming widely used as a new predictive tool in early drug discovery. The use of 3D cell cultures is believed to provide a more physiologically relevant response than monolayer (2D) cell cultures because they closely mimic the extracellular matrix and cell-cell interactions that occur in vivo.

The drug discovery and development pipeline are retooling imaging technologies, such as high content analysis, to accommodate 3D cell cultures as the model of choice.

In a high-content assay, subcellular organelles are analysed by high-resolution images captured by automated microscopes. High content imaging and analysis of 3D cell cultures provides:

  • Better predictive information on drug sensitivity
  • Improved drug-target validation
  • More accurate morphological and functional differentiation

However, evaluating 3D cell cultures comes with many limitations. Some of the factors that affect the quality of a 3D cell-based assay outcomes include a poor compound or fluorophore penetration, light penetration into or out of the spheroid/ organoid core, light scattering and cross-talk between channels when multiple fluorophores are excited simultaneously. Therefore, applying a combination of tools for assay preparation, image acquisition, visualisation, and data analysis to optimise the assay protocols are very important.

While there are various methods to evaluate 3D cell cultures, imaging technologies have advantages over other analytical methods. Imaging analysis enables multiple readouts without disrupting the sample physiology. Low-resolution microscopy is typically employed to track overall changes in spheroid structure and size. High-resolution microscopy using fluorescent probes provides data on individual cell structure, function, relative location, overall changes in the morphology and growth of the 3D structure and mechanisms underlying cellular responses to drugs. Current techniques for 3D fluorescence microscopy acquisition include widefield, confocal and super-resolution microscopy. Because of the thickness of the 3D spheroid samples, confocal microscopy can provide better background rejection and sharper images. For drug discovery, 3D imaging systems should be scalable, relatively high-throughput, fast, simple, reproducible and automation compatible.