The realization of soft robotic devices with life-like properties requires the engineering of smart, active materials that can respond to environmental cues in similar ways as living cells or organisms. Cell-free expression systems provide an approach for embedding dynamic molecular control into such materials that avoids many of the complexities associated with genuinely living systems. Here, we present a strategy to integrate cell-free protein synthesis within agarose-based hydrogels that can be spatially organized and supplied by a synthetic vasculature. We first utilize an indirect printing approach with a commercial bioprinter and Pluronic F-127 as a fugitive ink to define fluidic channel structures within the hydrogels. We then investigate the impact of the gel matrix on the expression of proteins in E. coli cell-extract, which is found to depend on the gel density and the dilution of the expression system. When supplying the vascularized hydrogels with reactants, larger components such as DNA plasmids are confined to the channels or immobilized in the gels while nanoscale reaction components can diffusively spread within the gel. Using a single supply channel, we demonstrate different spatial protein concentration profiles emerging from different cell-free gene circuits comprising production, gene activation, and negative feedback. Variation of the channel design allows the creation of specific concentration profiles such as a long-term stable gradient or the homogeneous supply of a hydrogel with proteins.

The growing need for sustainable biotechnological solutions to address environmental challenges, such as climate change and resource depletion, has intensified interest in microbial-based production systems. Synthetic biofilms, which mimic natural microbial consortia, offer a promising platform for optimizing complex metabolic processes that can convert renewable feedstocks into valuable chemicals. In this context, understanding and harnessing the interactions between co-immobilized microorganisms are critical for advancing bioprocesses that contribute to circular bioeconomy goals. In this study, we investigated the viability and metabolic activity of Clostridium carboxidivorans and Clostridium kluyveri within a synthetic, dual-layered biofilm composed of agar hydrogel. This setup compartmentalized each bacterial species. Embedding the bacteria in a structured biofilm offers numerous opportunities for bioproduction, but the inability to monitor cell growth or movement within the immobilization matrix limits process insights. To address this, we adapted a fluorescence in situ hybridization (FISH) protocol, enabling precise, species-specific visualization of bacterial distribution and growth within the gel matrix. Batch processes with the dual-layered biofilm in anaerobic flasks, designed with a metabolic advantage for C. kluyveri, revealed distinct growth dynamics. C. kluyveri exhibited significant metabolic activity, forming clusters at low initial cell concentrations and converting ethanol and acetate into 1-butyrate and 1-hexanoate, indicating viability and cell growth. C. carboxidivorans remained evenly distributed without significant growth or product formation, suggesting that while the cells were viable, they were not metabolically active under the experimental conditions. Both bacterial species were confined to their respective compartments throughout the process, with C. kluyveri showing enhanced substrate conversion at higher initial cell densities in the hydrogel. The pH drop throughout the batch experiment likely contributed to incomplete substrate consumption, particularly for C. kluyveri, which thrives within a narrow pH range. These findings highlight synthetic biofilms as a promising platform for optimizing microbial interactions and improving bioprocess efficiency, especially in applications involving complex metabolic exchanges between co-immobilized microorganisms. Further research will focus on applying conditions to support the growth and metabolic activity of C. carboxidivorans to explore spatial dynamics of bacterial migration and cooperative relationships in the synthetic biofilm.

Introducing subcellular structures in artificial cells is a key step in mimicking the structure and role of organelles, which are instrumental in compartmentalizing cellular reaction networks. Despite the variety of strategies to include subcellular features within artificial cell models, achieving spatial and morphological control over these compartments remains challenging. In this study, we engineered 3D-printed subcellular compartments within terpolymer-stabilized coacervate-based artificial cells. Coacervate-forming charged polymers were functionalized with methacrylate moieties, enabling the fabrication of a variety of architectures within droplets through photoinitiated radical polymerization. The addition of a Ni-NTA functional methacrylate monomer to the coacervates led to its sequestration upon polymerization in these subcellular regions. As a result, the compartments were able to uptake and concentrate His₆-tagged mTurquoise and β-galactosidase protein cargo molecules, despite the increase in viscosity that was induced upon polymerization. Following this affinity-based interaction approach, we demonstrated the region-specific localization of an enzymatic reaction within the artificial cells.

Engineering functional 3D tissue constructs is essential for developing advanced organ‐like systems, with applications ranging from fundamental biological research to drug testing. The generation of complex multicellular structures requires the integration of external geometric and mechanical cues with the ability to activate genetic programs that regulate and stimulate cellular self‐organization. Here, it is demonstrated that gelatin methacryloyl (GelMA) hydrogels serve as effective matrices for 3D cell culture, supporting both in situ genetic manipulation and cell growth. HEK293T cells embedded in GelMA remained viable and proliferated over 16 days, forming clusters within the matrix. Efficient gene delivery is achieved in the 3D hydrogel environment using both plasmid DNA and mRNA as gene vectors. Furthermore, in situ prime editing is applied to induce permanent genetic modifications in embedded cells. To achieve spatially confined gene expression, gel‐embedded channels are introduced that allowed localized stimulation via doxycycline perfusion through a Tet‐On system. These findings demonstrate the feasibility of integrating gene delivery, inducible expression, and spatial control within GelMA‐based hydrogels, establishing a versatile framework for engineered 3D cell systems with programmable genetic activity.

Engineering functional 3D tissue constructs is essential for developing advanced organ-like systems, with applications ranging from fundamental biological research to drug testing. The generation of complex multicellular structures requires the integration of external geometric and mechanical cues with the ability to activate genetic programs that regulate and stimulate cellular self-organization. Here, we demonstrate that gelatin methacryloyl (GelMA) hydrogels serve as effective matrices for 3D cell culture, supporting both in situ genetic manipulation and cell growth. HEK293T cells embedded in GelMA remained viable and proliferated over 16 days, forming clusters within the matrix. We achieved efficient gene delivery in the 3D hydrogel environment using both plasmid DNA and mRNA as gene vectors. Furthermore, we applied in situ prime editing to induce permanent genetic modifications in embedded cells. To achieve spatially confined gene expression, we introduced gel-embedded channels that allowed localized stimulation via doxycycline perfusion through a Tet-On system. Our findings establish GelMA hydrogel matrices as a versatile platform for generating spheroidal cell cultures while enabling precise genetic control and spatially resolved cellular manipulation through diffusible cues.