Additive Manufacturing of Metabolically Active Living Materials with Enhanced Biocontainment.

Additive manufacturing of engineered living materials (ELMs) enables the spatial patterning of living cells or organisms within three-dimensional (3D) polymer matrices. However, effective biocontainment, defined as the ability to retain cells within 3D-printed matrices during the application period, remains a key limitation for ELMs requiring physical confinement. While synthetic biology provides cell-specific biocontainment strategies, materials-based approaches can enable generic cell retention capabilities. Therefore, we applied a materials-based strategy that combines pre-printing formulation development and post-printing processing to increase the retention of yeast Saccharomyces cerevisiae in Pluronic F127-bisurethane methacrylate (F127-BUM) hydrogels. F127-BUM precursor solutions were formulated with the comonomers methacrylic acid (MAA) and/or 2-(dimethylamino)ethyl methacrylate (DMAEMA), extrusion-printed, UV-cross-linked, and immersed in 70 vol % ethanol for 60 or 300 s before cultivation. Comonomer addition preserved dimensional fidelity, thermoresponsiveness, and shear-thinning behavior for 3D printing. After immobilization and cross-linking, DMAEMA and higher concentrations of MAA considerably reduced yeast viability, whereas a 70-80% viability was observed for 0.03 and 0.06 mol L-1 MAA. With 0.06 mol L-1 MAA and 300 s ethanol immersion, the yeast retention time reached 98 ± 12 h, which was approximately 48% longer than that of the correspondingly treated F127-BUM-only control and represented about a 465% increase relative to the untreated F127-BUM-only control. After ethanol immersion, a peripheral growth-free zone developed around a metabolically active core, enhancing cell retention and contributing to delayed cell escape. The selected MAA-containing ELMs remained metabolically active and showed nearly 38% higher apparent glucose-to-ethanol conversion than treated F127-BUM-only controls. The suspension cultures fermented faster and achieved higher yield. Our method achieved enhanced biocontainment, with an operational window of about 4-5 days. In conclusion, we demonstrate that combining pre-printing and post-printing processes can enhance biocontainment for different ELM applications. We suggest the integration of this method with synthetic-biology strategies to further extend biocontainment duration for metabolically active living materials.