Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae

Background: Engineered living materials (ELMs) combine living cells with non-living scaffolds to obtain life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs can be 3D-printed and are called bioinks, and their scaffolds are mostly hydrogel-based. One such scaffold is polymer Pluronic F127, a liquid at 4 °C but a biocompatible hydrogel at room temperature. In such thermally-reversible hydrogel, the microorganism-hydrogel interactions remain uncharacterized, making truly durable 3D-bioprinted ELMs elusive. Methods: We demonstrate the methodology to assess cell-scaffold interactions by characterizing intact alive yeast cells in cross-linked F127-based hydrogels, using genetically encoded ratiometric biosensors to measure intracellular ATP and cytosolic pH at a single-cell level through confocal imaging. Results: When embedded in hydrogel, cells were ATP-rich, in exponential or stationary phase, and assembled into microcolonies, which sometimes merged into larger superstructures. The hydrogels supported (micro)aerobic conditions and induced a nutrient gradient that limited microcolony size. External compounds could diffuse at least 2.7 mm into the hydrogels, although for optimal yeast growth bioprinted structures should be thinner than 0.6 mm. Moreover, the hydrogels could carry whole-cell copper biosensors, shielding them from contaminations and providing them with nutrients. Conclusions: F127-based hydrogels are promising scaffolds for 3D-bioprinted ELMs, supporting a heterogeneous cell population primarily shaped by nutrient availability. Graphical Abstract: [Figure not available: see fulltext.]