To predict drug response or toxicity, the pharmaceutical industry is increasingly performing smaller-scale validation studies of experimental drug compounds in novel 3D cell culture models intended to mimic more closely the structure, activity, and extracellular environment of tissues in vivo.
Following high-throughput, large-scale screening assays performed in 2D systems, these smaller, secondary screens in 3D microtissue models are more likely to be predictive of how cells will react in vivo. Several presentations at SMi Group’s recent “Cell Culture” conference in London focused on advances in novel 3D cell culture systems and applications.
The things we see in 2D may not reflect what is happening in vivo, mainly due to the “lack of architecture in 2D systems, which influences the biological response in many difference phenotypes,” said Olivier Pardo, Ph.D., team leader at the Cellular Regulatory Networks Group, Imperial College, London. One clear example he cited is the response of tumor cells to drug therapy and the fact that tumor cells grown in 2D versus 3D cultures tend to respond quite differently to the same concentration of an antitumor drug. Tumor cells grown in 3D are typically more resistant to therapeutic compounds.
When cells grow in 3D spherical clusters, compared to 2D monolayers, the level of oxygenation differs depending on the whether a cell is situated more toward the inside or the outside of a spheroid structure. The difference in level of oxygenation appears to influence the response to drug therapy, explained Dr. Pardo. Additionally, differences in the extracellular matrix produced by cells growing in 3D culture systems can modify the cellular response to drugs and other stimuli, likely because of changes in integrin-based signaling in 3D compared to 2D cell cultures.
Another factor that can affect the therapeutic drug response of cells grown in culture is the extent of cell-to-cell contacts, which will tend to be more developed in 3D environments with higher cell densities.
3D culture systems are especially useful for studying the invasive properties and metastatic potential of tumor cells and for conducting screening assays for cell migration. In his presentation, Dr. Pardo described screens of large compound libraries using a bone metastatis assay or extra/intravasation assay, small-to-medium library screens with a 3D collagen invasion assay, and screening of a small number of targets using a zebrafish metastatic model organism. The 3D collagen invasion assay with confocal image acquisition was performed in 96-microwell plates.
Whereas manual screening was slow and difficult, making this assay format ill-suited for large library screening, the results obtained were highly reproducible, with >90% overlap between repeat results. In contrast, automating each step of the assay—and providing temperature control to minimize collagen polymerization—increased the throughput of the assay to make it suitable for large-scale screening, but the reproducibility of the robotized screen was poor, with <33% overlap between repeat results.
Limitations related to automation and the cost of most 3D cell culture technologies and materials available today are two important factors that stand in the way of using these methods for large-scale screening campaigns and of more widespread adoption of the technology, in Dr. Pardo’s view.
One of the main advantages of the 3D collagen assay and of 3D culture systems in general is the ability to do co-cultures, noted Dr. Pardo, thereby more closely mimicking the in vivo environment and enabling the introduction of cells capable of producing growth factors and other natural molecules to support the health and viability of the cultured cells.