Abstract text (incl. figure legends and references)
The microenvironment provides a vast array of signalling cues to cells that regulate cell behaviour and phenotype, all of which play a vital role in homeostasis. Given the complexity and ever-changing presentation of the extracellular matrix (ECM) in vivo, it remains challenging to effectively model and subsequently identify the primary drivers of cellular mechanosensitive responses in 3D. Furthermore, current biomaterial platforms such as hydrogels are coupled in the sense that to increase stiffness, porosity must be decreased, which may limit cell volume expansion. As such, this study aims to utilise tunable hydrogel platforms to draw conclusions about the independent effects of pore size and ECM stiffness on 3D stem cell mechanosensation. Human adipose-derived stem cells (ASCs) encapsulated in phototunable gelatin methacryloyl (GelMA) hydrogels were cultured at either a static or gradient stiffness, which aimed to achieve differential cell volume expansion. Following this, hydrogels were "on-demand" stiffened using UV photomasks to a desired stiffness and observations of cell morphology and protein expression were made. Cell volume was successfully limited using the phototunable hydrogel platform with cells being significantly larger in areas of lower hydrogel stiffness. Interestingly, in both platforms, a correlation between cell volume and nuclear mechanomarker localisation was observed. Similarly, stem cell differentiation showed a clear correlation with volume expansion, rather than the surrounding hydrogel stiffness. Local elasticity also appeared to be elevated in cells with larger volume expansion, which was quantified using optical coherence elastography (OCE). The current results from this platform suggest that 3D stem cell mechanosensation and differentiation are more closely correlated with volume expansion, rather than hydrogel stiffness. The findings may prove useful in controlling stem cells and biomaterial design for regenerative medicine purposes.