Complex diseases like cancer are more frequently being modelled using in vitro systems as more researchers opt for three-dimensional (3D) cell culture methods over two-dimensional (2D) ones. In some cases, 3D systems even have advantages over tumour xenograft models .
Recent advances in 3D cell culture techniques have made it possible to create environments that more closely resemble the native in vivo scenario in which cancers are found. The ability to co-culture multiple cell types that form higher-order structures, such as spheroids, recapitulates the tumour interior, where nutrient and hypoxia gradients persist.
At the other end of the spectrum, toward the tumour exterior, the capacity to supplement 3D cultures with extracellular matrix (ECM) proteins helps to recreate the structure, organization and functionality of live tissue in situ.
The benefits of studying cancer in 3D are manifold; so, if you’re looking to tailor a 3D culture environment to study a specific cancer of focus, but are not sure where to start, here are a few initial considerations to get your project moving…
Cell culture format
You need to consider which cell culture format is best for your experiment. Do you want to obtain spheroid cultures with intrinsic nutrient/oxygen/growth factor gradients that also secrete their own ECM proteins? If so, the hanging drop method of 3D cell culture is your best option. If you want to have greater control over ECM components for a better representation of the tumour stromal environment, then opt for a scaffold- or hydrogel-based culture medium.
Natural vs synthetic?
Gels of natural origin, such as collagen, and alginate, have been used for decades as substrates for 3D cell culture. The most commonly used gel among these is Matrigel™, a reconstituted basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. However, there are some concerns over the animal origins of natural gels and the effects they may have on cell cultures, for example they may contain residual growth factors, undefined constituents (such as animal viruses), or other nonquantified substances. Natural gels are also prone to batch-to-batch variations, rendering it difficult to compare work between different scientific groups.
Other concerns may stem from the distinctive qualities of the cancer growth environment. Despite Matrigel’s abundance in ECM proteins, such as laminin and certain collagen types, it does not contain high proportions of collagen type I or hyaluronan, which are found throughout the ECM of tumours in vivo at relatively high proportions .
To circumvent these issues, and bring about greater controllability over hydrogel composition, synthetic hydrogels have been developed. These can be modified to obtain desired characteristics through hybridization of natural and synthetic materials, incorporation of different types of proteins or molecules within the matrix, and hybridization of biomaterials with functional nanomaterials. The Dutch company Noviocell has even developed a biomimetic synthetic gel made up of a polymer called polyisocyanopeptide (PIC), the conformation of which closely resembles collagen, but retains all the reproducibility and controllability traits of a synthetic gel-based cell culture medium.
Specific ECM components and morphogenetic factors can be introduced into the PIC-based microenvironment to influence cancer cell growth and behaviour. The advances of PIC hydrogels will lead to a better understanding of the complex interactions of cancer cells with their extracellular microenvironment.
Once you have identified your culture strategy and cell growth medium, you need to decide what to supplement that with. This can include certain ECM components (proteins, glycoproteins, proteoglycans and polysaccharides), cytokines, specific growth factors or even intact cells. (See references 2 and 3 for more information.) Different functional groups (such as peptides containing the RDG sequence) can also be added to enhance cell-cell or cell-ECM interactions .
Multicellular co culture is also another avenue you could explore. Check the literature for any information on co culture of different cell types that will encourage more realistic growth conditions for your target cancer cell type.
One other thing you might want to consider is the density of your 3D cell culture medium. A major drawback of collagen-based hydrogels is that they contain a large amount of excess fluid (99%), and as such their very low density does not mimic the naturally stiff environment in which tumour cells (and most tissue cells) naturally reside. Synthetic scaffold and gel-based approaches are one way to obtain more control over culture density.
Synthetic 3D culture media have great potential to mimic aspects of the tumour microenvironment due to their easy functionalization and manipulation; in effect, they can be designed to requirements. Nonetheless, challenges remain to achieve a true in vivo-like tumour environment with all the essential cues required to mimic a cancer’s behavior, and importantly response to therapeutic agents. Hybrid 3D supports like the PIC Hydrogel are a welcome step towards this goal.
 Nyga A., Cheema U., Loizidou M. et al. 3D tumour models: novel in vitro approaches J. Cell Commun. Signal. (2011); 5:239-248.
 Cunliffe D, Pennadam S, Alexander C (2004) Synthetic and biological polymers—merging the interface. Eur Polym J 40:5–25
 Chen G, Sato T, Ushida T, Hirochika R, Shirasaki Y, Ochiai N, Tateishi T (2003) The use of a novel PLGA fiber/collagen 246 A. Nyga et al. composite web as a scaffold for engineering of articular cartilage tissue with adjustable thickness. J Biomed Mater Res A 67:1170– 1180
 Villanueva I, Weigel CA, Bryant SJ (2009) Cell-matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels. Acta Biomater 5:2832–2846