Enhancing long-term storage and stability of engineered living materials through desiccant storage and trehalose treatment.

Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.

3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix.

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. Bio-based materials that support cellular adhesion, differentiation, and proliferation - including gelatin, collagen, hyaluronic acid, and alginate - have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states. The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Additive Manufacturing of Bovine Serum Albumin-Based Hydrogels and Bioplastics.

Biosourced and biodegradable polymers for additive manufacturing could enable the rapid fabrication of parts for a broad spectrum of applications ranging from healthcare to aerospace. However, a limited number of these materials are suitable for vat photopolymerization processes. Herein, we report a two-step additive manufacturing process to fabricate robust protein-based constructs using a commercially available laser-based stereolithography printer. Methacrylated bovine serum albumin (MA-BSA) was synthesized and formulated into aqueous resins that were used to print complex three-dimensional (3D) objects with a resolution comparable to a commercially available resin. The MA-BSA resins were characterized by rheometry to determine the viscosity and the cure rate, as both parameters can ultimately be used to predict the printability of the resin. In the first step of patterning these materials, the MA-BSA resin was 3D printed, and in the second step, the printed construct was thermally cured to denature the globular protein and increase the intermolecular noncovalent interactions. Thus, the final 3D printed part was comprised of both chemical and physical cross-links. Compression studies of hydrated and dehydrated constructs demonstrated a broad range of compressive strengths and Young's moduli that could be further modulated by adjusting the type and amount of co-monomer. The printed hydrogel constructs demonstrated good cell viability (>95%) after a 21 day culture period. These MA-BSA resins are expected to be compatible with other vat photopolymerization techniques including digital light projection and continuous liquid interface production.

Additive Manufacturing of Catalytically Active Living Materials.

Living materials, which are composites of living cells residing in a polymeric matrix, are designed to utilize the innate functionalities of the cells to address a broad range of applications such as fermentation and biosensing. Herein, we demonstrate the additive manufacturing of catalytically active living materials (AMCALM) for continuous fermentation. A multi-stimuli-responsive yeast-laden hydrogel ink, based on F127-dimethacrylate, was developed and printed using a direct-write 3D printer. The reversible stimuli-responsive behaviors of the polymer hydrogel inks to temperature and pressure are critical, as they enabled the facile incorporation of yeast cells and subsequent fabrication of 3D lattice constructs. Subsequent photo-cross-linking of the printed polymer hydrogel afforded a robust elastic material. These yeast-laden living materials were metabolically active in the fermentation of glucose into ethanol for 2 weeks in a continuous batch process without significant reduction in efficiency (∼90% yield of ethanol). This cell immobilization platform may potentially be applicable toward other genetically modified yeast strains to produce other high-value chemicals in a continuous biofermentation process.

Catalytically Initiated Gel-in-Gel Printing of Composite Hydrogels.

Herein, we describe a method to 3D print robust hydrogels and hydrogel composites via gel-in-gel 3D printing with catalytically activated polymerization to induce cross-linking. A polymerizable shear-thinning hydrogel ink with tetramethylethylenediamine as catalyst was directly extruded into a shear-thinning hydrogel support bath with ammonium persulfate as initiator in a pattern-wise manner. When the two gels came into contact, the free radicals generated by the catalyst initiated the free-radical polymerization of the hydrogel ink. Unlike photocuring, a catalyst-initiated polymerization is suitable for printing hydrogel composites of varying opacity, since it does not depend upon light penetration through the sample. The hydrogel support bath also exhibited a temperature-responsive behavior in which the gel "melted" upon cooling below 16 °C. Therefore, the printed object was easily removed by cooling the gel to a liquid state. Hydrogel composites with graphene oxide and multiwalled carbon nanotubes (MWCNTs) were successfully printed. The printed composites with MWCNTs afforded photothermally active objects, which have utility as stimuli-responsive actuators.