Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, an understanding of the cellular phenotype within living materials is lacking, which is a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photo-cross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

Living materials are created through the embedding of live, whole cells into a matrix that can house and sustain the viability of the encapsulated cells. Through the immobilization of these cells, their bioactivity can be harnessed for applications such as bioreactors for the production of high‐value chemicals. While the interest in living materials is growing, many existing materials lack robust structure and are difficult to pattern. Furthermore, many living materials employ only one type of microorganism, or microbial consortia with little control over the arrangement of the various cell types. In this work, a Pluronic F127‐based hydrogel system is characterized for the encapsulation of algae, yeast, and bacteria to create living materials. This hydrogel system is also demonstrated to be an excellent material for additive manufacturing in the form of direct write 3D‐printing to spatially arrange the cells within a single printed construct. These living materials allow for the development of incredibly complex, immobilized consortia, and the results detailed herein further enhance the understanding of how cells behave within living material matrices. The utilization of these materials allows for interesting applications of multikingdom microbial cultures in immobilized bioreactor or biosensing technologies.

Smart and soft electroactive polymer actuators have many beneficial properties, making them attractive for biomimetic and biomedical applications. However, the selection of components to fabricate biofriendly composites has been limited. Although biofriendly options for electrodes and membranes are available, the conventional ionic liquids (ILs) often used as the electrolytes in the actuators have been considered toxic in varying degrees. Here we present a smart electroactive composite with carefully designed and selected components that have shown low toxicity and a biofriendly nature. In the present study, polypyrrole-PVdF trilayer actuators using six different choline ILs were prepared and characterized. Choline ILs have shown promise in applications where low environmental and biological impact is critical. Despite this, the anions in ILs have a strong impact on toxicity. To evaluate how the anions effect the bioactivity of the ILs used to prepare the actuators, the ILs were tested on different microbial cultures (Escherichia coli, Staphylococcus aureus, Shewanella oneidensis MR-1) and HeLa cells. All of the selected choline ILs showed minimal toxic effects even at high concentrations. Electro-chemo-mechanical characterization of the actuators indicated that polypyrrole-PVdF actuators with choline ILs are viable candidates for soft robotic applications. From the tested ILs, choline acetate showed the highest strain difference and outperformed the reference system containing an imidazolium-based IL.

The three-dimensional (3D) printing of cell-containing polymeric hydrogels creates living materials (LMs), offering a platform for developing innovative technologies in areas like biosensors and biomanufacturing. The polymer material properties of cross-linkable F127-bis-urethane methacrylate (F127-BUM) allow reproducible 3D printing and stability in physiological conditions, making it suitable for fabricating LMs. Though F127-BUM-based LMs permit diffusion of solute molecules like glucose and ethanol, it remains unknown whether these are permissible for oxygen, essential for respiration. To determine oxygen permissibility, we quantified dissolved oxygen consumption by the budding yeast-laden F127-BUM-based LMs. Moreover, we obtained data on cell-retaining LMs, which allowed a direct comparison between LMs and suspension cultures. We further developed a highly reliable method to isolate cells from LMs for flow cytometry analysis, cell viability evaluation, and the purification of macromolecules. We found oxygen consumption heavily impaired inside LMs, indicating that yeast metabolism relies primarily on fermentation instead of respiration. Applying this finding to brewing, we observed a higher (3.7%) ethanol production using LMs than the traditional brewing process, indicating improved fermentation. Our study concludes that the present F127-BUM-based LMs are useful for microaerobic processes but developing aerobic bioprocesses will require further research.

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