Scalable Processes for Culturing Meat Using Edible Scaffolds.

There is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that both is palatable and has reduced environmental impact. However, strategies to support the production of cultured meats at the scale required for food consumption will be critical. In this review, we discuss the current challenges and opportunities of using edible scaffolds for scaling up the production of cultured meat. We provide an overview of different types of edible scaffolds, scaffold fabrication techniques, and common scaffold materials. Finally, we highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat.

Engineering multicomponent tissue by spontaneous adhesion of myogenic and adipogenic microtissues cultured with customized scaffolds.

The integration of intramuscular fat-or marbling-into cultured meat will be critical for meat texture, mouthfeel, flavor, and thus consumer appeal. However, culturing muscle tissue with marbling is challenging since myocytes and adipocytes have different media and scaffold requirements for optimal growth and differentiation. Here, we present an approach to engineer multicomponent tissue using myogenic and adipogenic microtissues. The key innovation in our approach is the engineering of myogenic and adipogenic microtissues using scaffolds with customized physical properties; we use these microtissues as building blocks that spontaneously adhere to produce multicomponent tissue, or marbled cultured meat. Myocytes are grown and differentiated on gelatin nanofiber scaffolds with aligned topology that mimic the aligned structure of skeletal muscle and promotes the formation of myotubes in both primary rabbit skeletal muscle and murine C2C12 cells. Pre-adipocytes are cultured and differentiated on edible gelatin microbead scaffolds, which are customized to have a physiologically-relevant stiffness, and promote lipid accumulation in both primary rabbit and murine 3T3-L1 pre-adipocytes. After harvesting and stacking the individual myogenic and adipogenic microtissues, we find that the resultant multicomponent tissues adhere into intact structures within 6-12 h in culture. The resultant multicomponent 3D tissue constructs show behavior of a solid material with a Young's modulus of âˆ¼ 2 ± 0.4 kPa and an ultimate tensile strength of âˆ¼ 23 ± 7 kPa without the use of additional crosslinkers. Using this approach, we generate marbled cultured meat with âˆ¼ mm to âˆ¼ cm thickness, which has a protein content of âˆ¼ 4 ± 2 g/100 g that is comparable to a conventionally produced Wagyu steak with a protein content of âˆ¼ 9 ± 4 g/100 g. We show the translatability of this layer-by-layer assembly approach for microtissues across primary rabbit cells, murine cell lines, as well as for gelatin and plant-based scaffolds, which demonstrates a strategy to generate edible marbled meats derived from different species and scaffold materials.

Effects of trehalose and polyacrylate-based hydrogels on tomato growth under drought.

Hydrophilic amendments can enhance soil moisture content, which, in turn, can improve crop health under drought conditions. Understanding how different hydrogels interact with specific crops is necessary for optimal application. The soil conditioning abilities of a trehalose hydrogel and polyacrylate-based hydrogel were evaluated for tomatoes (Solanum lycopersicum) subjected to drought. Tomato plants were transplanted into individual pots with soil that contained trehalose hydrogel (0.4 wt%), polyacrylate-based hydrogel (0.4 wt%), or no hydrogel and subjected to a well-watered treatment or to pronounced soil drought, with or without rewatering. The health of tomato plants was monitored by measuring leaf total chlorophyll (a + b) concentration, leaf water potential (Ψleaf), stomatal conductance (g s) and relative growth rate (RGR). The polyacrylate-based hydrogel, but not the trehalose hydrogel, improved tomato plant function under drought conditions, as indicated by improved g s and RGR relative to the well-watered control. However, when subjected to a second drought, neither hydrogel was effective, and neither prolonged survival. The more hydrophilic polyacrylate-based hydrogel demonstrated promise in improving the growth of tomato plants under drought when included as a soil amendment at 0.4 wt%. This research is important for understanding the effects of these hydrogels as soil conditioners in drought prone systems.

Emulsion-templated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat.

Cultured meat has potential to diversify methods for protein production, but innovations in production efficiency will be required to make cultured meat a feasible protein alternative. Microcarriers provide a strategy to culture sufficient volumes of adherent cells in a bioreactor that are required for meat products. However, cell culture on inedible microcarriers involves extra downstream processing to dissociate cells prior to consumption. Here, we present edible microcarriers that can support the expansion and differentiation of myogenic cells in a single bioreactor system. To fabricate edible microcarriers with a scalable process, we used water-in-oil emulsions as templates for gelatin microparticles. We also developed a novel embossing technique to imprint edible microcarriers with grooved topology in order to test if microcarriers with striated surface texture can promote myoblast proliferation and differentiation in suspension culture. In this proof-of-concept demonstration, we showed that edible microcarriers with both smooth and grooved surface topologies supported the proliferation and differentiation of mouse myogenic C2C12 cells in a suspension culture. The grooved edible microcarriers showed a modest increase in the proliferation and alignment of myogenic cells compared to cells cultured on smooth, spherical microcarriers. During the expansion phase, we also observed the formation of cell-microcarrier aggregates or 'microtissues' for cells cultured on both smooth and grooved microcarriers. Myogenic microtissues cultured with smooth and grooved microcarriers showed similar characteristics in terms of myotube length, myotube volume fraction, and expression of myogenic markers. To establish feasibility of edible microcarriers for cultured meat, we showed that edible microcarriers supported the production of myogenic microtissue from C2C12 or bovine satellite muscle cells, which we harvested by centrifugation into a cookable meat patty that maintained its shape and exhibited browning during cooking. These findings demonstrate the potential of edible microcarriers for the scalable production of cultured meat in a single bioreactor.

Bottlebrush polymers with flexible enantiomeric side chains display differential biological properties.

Chirality and molecular conformation are central components of life: biological systems rely on stereospecific interactions between discrete (macro)molecular conformers, and the impacts of stereochemistry and rigidity on the properties of small molecules and biomacromolecules have been intensively studied. Nevertheless, how these features affect the properties of synthetic macromolecules has received comparably little attention. Here we leverage iterative exponential growth and ring-opening metathesis polymerization to produce water-soluble, chiral bottlebrush polymers (CBPs) from two enantiomeric pairs of macromonomers of differing rigidity. Remarkably, CBPs with conformationally flexible, mirror image side chains show several-fold differences in cytotoxicity, cell uptake, blood pharmacokinetics and liver clearance; CBPs with comparably rigid, mirror image side chains show no differences. These observations are rationalized with a simple model that correlates greater conformational freedom with enhanced chiral recognition. Altogether, this work provides routes to the synthesis of chiral nanostructured polymers and suggests key roles for stereochemistry and conformational rigidity in the design of future biomaterials.

Synthetic Glycomacromolecules of Defined Valency, Absolute Configuration, and Topology Distinguish between Human Lectins.

Carbohydrate-binding proteins (lectins) play vital roles in cell recognition and signaling, including pathogen binding and innate immunity. Thus, targeting lectins, especially those on the surface of immune cells, could advance immunology and drug discovery. Lectins are typically oligomeric; therefore, many of the most potent ligands are multivalent. An effective strategy for lectin targeting is to display multiple copies of a single glycan epitope on a polymer backbone; however, a drawback to such multivalent ligands is they cannot distinguish between lectins that share monosaccharide binding selectivity (e.g., mannose-binding lectins) as they often lack molecular precision. Here, we describe the development of an iterative exponential growth (IEG) synthetic strategy that enables facile access to synthetic glycomacromolecules with precisely defined and tunable sizes up to 22.5 kDa, compositions, topologies, and absolute configurations. Twelve discrete mannosylated "glyco-IEGmers" are synthesized and screened for binding to a panel of mannoside-binding immune lectins (DC-SIGN, DC-SIGNR, MBL, SP-D, langerin, dectin-2, mincle, and DEC-205). In many cases, the glyco-IEGmers had distinct length, stereochemistry, and topology-dependent lectin-binding preferences. To understand these differences, we used molecular dynamics and density functional theory simulations of octameric glyco-IEGmers, which revealed dramatic effects of glyco-IEGmer stereochemistry and topology on solution structure and reveal an interplay between conformational diversity and chiral recognition in selective lectin binding. Ligand function also could be controlled by chemical substitution: by tuning the side chains of glyco-IEGmers that bind DC-SIGN, we could alter their cellular trafficking through alteration of their aggregation state. These results highlight the power of precision synthetic oligomer/polymer synthesis for selective biological targeting, motivating the development of next-generation glycomacromolecules tailored for specific immunological or other therapeutic applications.