“Smart” materials are substances designed to be responsive to environmental stimuli, e.g., to changes in temperature. One class of such thermoresponsive polymers is Pluronic F127, a biologically compatible yet reversely-gelling material, i.e., one liquid at near 0°C but hydrogel at room temperature. Because of this property, it can be mixed with cells on ice and then 3D-printed at room temperature, thus serving as a convenient bioink matrix. Ends of Pluronic F127 molecules can also be chemically modified to support the fixation of hydrogel into one shape, no matter the temperature. However, beyond rudimentary observations of biocompatibility, it remains unclear how cells cope with conditions inside such a substance. To investigate this issue, we immobilized yeast S. cerevisiae in Pluronic F127-dimethacylate (F127-DMA), molded the produced hydrogel, fixed its shape through photopolymerization, and incubated it in either water or yeast synthetic medium. To investigate physiology and energetics at a single-cell level, we applied a genetically encoded GFP-based ratiometric biosensor which allows for the measurement of physiologically relevant concentrations of intracellular ATP. Our results confirm the biocompatibility of F127-DMA but also show it promotes the development of very heterogeneous cell populations that reflect nutrient and oxygen gradients pervading the hydrogel. Thus, cells near the edge of the hydrogel proliferate quickly and even form microcolonies. In contrast, those removed from the edge divide infrequently and are seemingly dormant, demonstrating that in bioink and 3D printing, the location does matter.