3D cell culture: Another dimension to drug discovery

Cell biologists are slowly replacing two-dimensional (2D) cell culture with more advanced alternatives, such as growing cells in three-dimensions (3D) by their encapsulation in hydrogels. Unlike 2D cell culture, where cells adhere to the culture vessel’s flat, hard surfaces in monolayers, 3D systems facilitate a higher order of cellular spatial organization and thus yield more physiologically relevant information – a definite advantage if you are looking to put a potential therapeutic molecule through its paces.

Superior translatability 

It is now generally accepted that cells behave differently in 3D environments compared to 2D ones, particularly when it comes to drug discovery – many potential cancer therapeutics look promising in the 2D cell culture dish, but fall woefully short of their main clinical goals later on in clinical development [1]. This undesirable outcome may be down to an observation made by several independent studies: that tumor cells are more resistant to anti-cancer agents when evaluated in a 3D systems compared to 2D culture conditions [2-4]. The cause of this is a culmination of the following common characteristics of 3D cell culture environments:

  • Presence of oxygen and nutrient gradients – cultures can be used as efficient simulators of tumor characteristics such hypoxia.
  • Increased cell-to-cell interactions because of cellular 3D architecture – proximity of other cells on all sides is more akin to in vivo conditions.
  • Non-uniform drug/compound exposure – a thick layer of polymeric 3D molecules (particularly spheroid cultures) provides a better representation of tissue penetration by a drug candidate in vivo.
  • Capacity for ECM-to-cell signaling – 3D cell culture environments, specifically hydrogels, can be engineered to present a more realistic microenvironment to cells, for example they can be augmented with ECM components.
  • Alterations in gene expression patterns and cell behavior in 3D versus 2D environments. Genetic alterations of interest included a number of chemokines (CXCL1, CXCL2 and CXCL3), and IL-8 and CCL20, which were significantly up-regulated in cells cultured in 3D conditions [5,6].

Current drawbacks

At present, there are a number of commercially available materials suitable for 3D cell culture, which can be subdivided into two groups: natural and synthetic hydrogels. Natural hydrogels (e.g. Matrigel®) are extracted from biological sources (e.g. mouse sarcoma) and can be laminin-, collagen-, alginate- and hyaluronic acid-based, or a mixture of several of these. They may also contain various growth factors. Currently they remain the most popular choice of growth medium in 3D cell culture; however, those wishing to utilize their benefits in drug discovery need to take into consideration that natural 3D cell culture media have their drawbacks. For instance, they suffer from batch-to-batch variation: this is because their exact composition cannot be defined due to their origin and, consequently, the responses of the cells embedded are difficult to control affecting the reproducibility of experimental results.

Other drawbacks of natural 3D media include:

  • Pathogen transmission and immunogenicity, due to their origin; 
  • Technical challenges in handling and with downstream processing after culturing: difficulties in cell or organoid isolation, not suitable for imaging with confocal microscopy, difficulties with antibody staining;
  • Due to their composition, they cannot be used for regenerative medicine. 

In response to this situation, several companies have developed synthetic hydrogels as alternatives to natural 3D culture gels. However, these do not adequately represent the complicated ECM that surrounds cells in tissues, and thus they often fail to simulate essential biological interactions.

The best of both worlds

Noviocell has developed a fully synthetic biomimetic extracellular matrix, called polyisocyanopeptide (PIC) hydrogel, that has almost identical biomechanical properties to natural matrices. However, due to PIC’s synthetic nature, it forms a gel of uniform composition, by-passing the batch-to-batch variation issues of natural gels.This constant composition is easier to manipulate in comparison to natural hydrogels, and characteristics such as nutrient, gas and drug diffusion rates, cell shear stresses and other microenvironmental conditions can be more accurately controlled with the PIC system, making it an excellent matrix for 3D cell culture applications, including the initial stages of drug selection. 

References

[1] News GEaB. Top 10 Clinical Trial Failures of 2013. www.genengnews.com/insight-and-intelligenceand153/top-10-clinical-trial-failures-of-2013/77900029

[2] Dhiman H.K., Ray A.R., Panda A.K. Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials. 2005;26:979–986. doi: 10.1016/j.biomaterials.2004.04.012.

[3] Horning J.L., Sahoo S.K., Vijayaraghavalu S., Dimitrijevic S., Vasir J.K., Jain T.K., Panda A.K., Labhasetwar V. 3-d tumor model for in vitro evaluation of anticancer drugs. Mol. Pharm. 2008;5:849–862. doi: 10.1021/mp800047v. 

[4] Barbone D., Yang T.M., Morgan J.R., Gaudino G., Broaddus V.C. Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids. J. Biol. Chem. 2008;283:13021–13030. doi: 10.1074/jbc.M709698200. 

[5] Ghosh S., Spagnoli G.C., Martin I., Ploegert S., Demougin P., Heberer M., Reschner A. Three-dimensional culture of melanoma cells profoundly affects gene expression profile: A high density oligonucleotide array study. J. Cell Physiol. 2005;204:522–531. doi: 10.1002/jcp.20320. 

[6] Lee J.M., Mhawech-Fauceglia P., Lee N., Parsanian L.C., Lin Y.G., Gayther S.A., Lawrenson K. A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro. Lab Invest. 2013;93:528–542. doi: 10.1038/labinvest.2013.41.