1. Facilitate the understanding of structure–function relationship in normal and pathological tissue conditions. Studies have shown that human annulus disc cells cultured in 3 dimensional gel systems showed different morphology than those cultured in 2D. These cells cultured in 3D showed increased proteoglycan synthesis compared to monolayer grown cells, and formation of multi-celled colonies with extracellular matrix deposited around and between cells. Furthermore, the human annulus disc cells cultured in 3 dimensional showed the evidence of Type I and II collagen production which was not found in mono-layer cell culture.

In vitro animal cell growth in 3D promotes normal epithelial polarity and differentiation. Cells move and divide more quickly and have a characteristically asymmetric shape compared with that of cells in living tissue.

2. 3D cell culture is a better model for studying the interactions between cell and growth factors as well as cell and therapeutic agents.  For example, a three dimensional cell culture of cancer cells allows the exploration of many basic questions related to cancer biology, as receptors for tumor development growth factors are expressed in different ways in comparison to the standard 2 dimensional tissue culture plates. For breast cancer, 3 dimensional culture provides a model system for understanding the regulation of cancer cell proliferation and for evaluation of different anticancer drugs. There is a substantial amount of evidence that cells growing in 3D culture are more resistant to cytotoxic agents than cells in monolayer or dispersed culture. Many studies have demonstrated an elevated level of drug resistance of spheroids culture compared with cells in monolayer. Initially, investigators attributed drug resistance of spheroids to poor diffusion of the drugs to interior cells but now it has been proved that only 3 dimensional culture accounts for drug resistance rather than mere inaccessibility to nutrients. Further study confirmed that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.

3. 3D cell culture systems in vitro can facilitate the understanding of structure–function relationship in normal and pathological conditions. It is now well accepted that bone and cartilage-derived cells behave differently in 3D than in a 2D environment and that the 3D culture systems in vitro are mimicking the in vivo situation more closely than the 2D cultures. In a recent study, three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D hydrogel matrix. It was demonstrated that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines indicate that all the cells cultured in 3D in this hydrogel matrix exhibited a more mature differentiation status than cells cultured in monolayer on plastic cell culture plates.

4. 3D culture is a better technique for stem cell expansion. Human Embryonic Stem Cells (hESC), when culture in 3D for expansion, showed a much higher cell number after 30 day expansion, compared to 2D expansion in cell culture plate. Expanded hESC are able to differentiate into representatives of the three germ layers: ectoderm, endoderm, and mesoderm.