Angiogenesis is recurrent function among the genes listed in Table 3, which is not surprising. As already mentioned, as a microtissue grows beyond a certain size, nutrient and oxygen depletion become limiting factors leading to the inhibition of cell proliferation and initiation of Enzalutamide 915087-33-1 angiogenic signaling. Oxygen concentration in 3D tissues depends on the balance between oxygen delivery and consumption. In vivo, this balance is tightly regulated by evenly distributed capillary networks but in vitro homotypic 3D microtissues lack vasculature and therefore develop a hypoxic core as their size increases. This event leads to the cells producing chemical signals for angiogenesis and is quite similar to the response occurring in normal hypoxic tissues where balanced signaling cascades lead to vascular remodeling and angioadaptation until the tissue oxygen concentration is back within its normal range. The up-regulation of VEGF-A and other genes with angiogenic function like ANGPTL7/CDT6, FGF5, NOV/IBP-9, and TGFA is pointing to a functional class of cytokines with great potential as three-dimensionality biomarkers. The above genes may play other roles besides being factors involved with hypoxia induced angiogenesis. For example, VEGFA has been shown to regulate neuronal development, survival, neurite growth and it also possesses gliotrophic properties. Also, it’s up-regulation is not limited to neuronal cells; it has been shown to be up-regulated in 3D cultures of a variety of cell and tissue types including human fetal lung fibroblasts, oral squamous cell carcinoma, glioblastoma, breast cancer, neonatal rat cardiomyocytes and neonatal mouse cardiomyocytes. This up-regulation in a non-cell type specific manner in 3D cultures of VEGF-A, and probably other members of the class, lends credibility to the notion of ubiquity of these potential biomarkers in different cells derived from different tissue types. Another recurrent gene function theme in Table 3 is terminal differentiation, a process by which cells commit to being part of a particular tissue or organ and perform a particular function. Terminal differentiation is preceded by inhibition of proliferation and cell cycle arrest. This exerts endoplasmic reticulum stress on the cell, but anti-apoptotic factors like Bcl-2 protect the cell from apoptosis and commit it to differentiation. In 3D, much like in organs in vivo, there is a spatial constraint on the microtissue, exerted by the defined pore size of the scaffold, which prevents them from proliferating freely, maintaining them in a quiescent state. Evidence in support of this comes from the fact that cells grown in 3D have shown lower proliferation rates than their 2D counterparts and higher expression of cyclin dependent kinase inhibitors p21. Also integrin mediated adhesion to the ECM leads to activation of Bcl-2 family of genes and makes the 3D cells more resistant to apoptosis. Such conditions are favorable for the cell to undergo terminal differentiation and this can be clearly seen by up-regulation in a number of differentiation and survival factor in 3D and NS compared to 2D, like PDGFB and STC1.