Although cytokines have been extensively studied in the field of immunology and oncology, tissue or cell-based biosensor engineers have paid little attention to these small proteins that have potential to revolutionize the field. The evidence for their existence in 3D cultures is compelling but they have not yet been looked at as candidates for potential 3D biomarkers. However, they were an ideal family to explore for the search. The rationale behind their choice was based on the fact that, in a 3D microenvironment cells are surrounded by homotypic neighbors forming a loosely bound disorganized aggregate. When compared to in vivo, such a scenario exists only during avascular tumorogenesis or early stages of inflammatory wound healing and both these phenomenon are regulated by the same molecules – Cytokines. So in vitro, the cells growing in 3D relate to any of those two models depending upon their type – malignant or primary, respectively, and therefore upregulation of their cytokine levels was physiologically relevant. Consistent with our results, Y-27632 dihydrochloride ROCK inhibitor up-regulation of cytokines in 3D cultures compared to 2D has been reported by several transcriptomic studies using cells from the four main tissue types cultured in a wide variety of platforms. For example, Klapperich and Bertozzi showed that seven cytokines were up-regulated in human fetal lung fibroblasts cultured in a collagen–glycosaminoglycan 3D mesh when compared to 2D surfaces. Also, upregulation of six cytokines by a melanoma cell line cultured on poly-2- hydroxyethyl methacrylate plates was reported by Ghosh at al.. Transcriptomic findings such as those in the above examples have been further substantiated by studies at the protein level. For example, Enzerink et al. have reported induction of chemokine secretion due to clustering of cells in five different fibroblast cell lines cultured in agarose. Also, Fischbach et al. cultured tumor cells in a 2D and 3D RGD-alginate system and reported a dramatic enhancement of IL-8 levels in 3D. Another study by the same group showed that when the same cells were grown in Matrigel there was up-regulation of cytokines when compared to 2D. This observation is of particular importance as cells grown on Matrigel have already been shown to produce an outcome similar to in vivo, like the formation of mammary gland acinus and milk-like secretion into lumen proving that Matrigel can provide all the relevant microenvironmental factors. This suggests that the up-regulation of cytokines in 3D compared to 2D is not a random differential response but is pertinent as a similar response is elicited when a proven physiologically relevant microenvironmental platform is provided. We have used the themes of tumorogenesis, inflammation and development as shown in Table 3 to closely examine the13 upregulated transcripts in both 3D and NS culture conditions; cells in a 3D culture in vitro relate to in vivo phenomenon like avascular tumor progression, early stages of inflammatory wound healing or embryonic development depending upon their type- malignant, primary or stem.