However, with recent advances in the field in the past decade, the meaning of 3D cell culture has been extended to providing the “total microenvironment” that supports the formation of microtissue that exhibits “complex” physiological relevance or better emulation of the in vivo functionality in a manner not possible in 2D cultures. Three main categories or microenvironment factors or “three-dimensions” from the literature include: 1) chemical or biochemical composition, 2) spatial and temporal dimensions, and 3) force and substrate physical properties. However, there is still a lack of a quantifiable entity which can establish if the cellular response in a 3D culture is actually physiologically relevant and in vivo-like or just different from 2D. The identification and validation for this entity or a potential three-dimensionality biomarker is necessary due to three compelling reasons. First, apart from the concept of “three-dimensional matrix adhesion” originally proposed by Cukierman et al. as a possible indication or “diagnosis” or marker for a culture state of three-dimensionality, the fields of tissue engineering and/or cell-based biosensors have not provided knowledge on the basis of which a consensus for threedimensionality and the associated complex physiological relevance could be established. Because of this, claims of “physiologically more relevant” are readily made for cells cultured on any surface or scaffold that provides loosely defined 3D geometry, either at the nano- or micro- structure levels or their combinations, as long as the resulting cell phenotypes are different between the 2D and 3D geometries. Second, the concept of using combinatorial approaches to fabricate libraries of polymers or other material scaffolds for tissue engineering or cell-based drug discovery call for high throughput assay by which “hit materials” can be quickly identified for further development. Cell-material interaction outcome can potentially guide the development of such assays or biosensors. An interaction with a material which yields cells that emulate in vivo conditions would be most desirable. Threedimensionality biomarkers would provide the intellectual basis for material discovery platform development. Third, in order to lower the costs associated with 3D platforms and make them more accessible for high throughput screening Life Science Reagents applications, simplification of the platform without giving up the physiologically relevant behavior of the cells is necessary, as discussed in detail by Lai et al.. Taken together, the subfield or field of 3D culture needs ubiquitous validated biomarkers. As a first step, in search for threedimensionality biomarkers, we initiated a cytokine expression comparative transcriptomic study with neural progenitor cells grown on 2D flat surfaces, 3D polymeric scaffolds and as neurospheres. NS were used as the in vivo surrogate, since they have been shown to emulate many in vivo functions that have not been possible in 2D cultures. Cytokines are involved in many crucial cell functions like innate and adaptive inflammatory host defenses, cell growth, differentiation, cell death, angiogenesis, and development and repair processes.