Speaker
Description
Introduction
With a growing world population, humanity faces increasing demands for food and availability of meat. Conventional mass production is inefficient, uses up valuable resources, and leads to detrimental ecological and ethical consequences. Cellular agriculture applies muscle tissue engineering principles to develop future protein sources. While proof-of-concept generation of cultured meat on a smaller scale has been reported, several challenges remain on the path towards biofabrication of whole cuts of beef. Here, we explore the design of sustainable, edible biomaterial inks for scalable bioprinting of nutritious engineered beef. Two major components of the extracellular matrix (ECM) in bovine muscle tissue are elastin and laminin, both of which help guide myogenic differentiation of bovine muscle precursor/satellite cells (bMuSC). To mimic these features in animal-free scaffolds, we designed a recombinant, engineered protein that includes peptide sequences from both elastin and laminin.[1,2] To formulate the engineered protein into an edible scaffold, we developed a vitamin-based strategy to achieve light-based curing of a protein matrix supporting muscle formation.
Materials and methods
An elastin-like protein (ELP) exhibiting a bioactive, cell-instructive, laminin-mimicking sequence was expressed in engineered microorganisms (Escherichia coli).[2] Artificial muscle samples were assembled by seeding or encapsulating C2C12 myoblasts or primary bMuSC into the ELP (2×107 cells per mL). Photo-crosslinking was initiated at 450 nm after riboflavin (vitamin B2) activation.[3] Rheological bioink properties with and without edible viscosity enhancers were investigated using a stress-controlled rheometer (ARG2, TA Instruments). Shear thinning behavior of inks was analyzed by flow curves (0.1-1000 s-1) and frequency sweeps (10-1-102 rad/s). Printability was evaluated after extrusion bioprinting of model test structures and of constructs with multiple, parallel, muscle-like bundles.
Cell survival, myotube formation, and contractility were characterized in serum-containing and serum-free culture conditions. Myogenic differentiation was evaluated by mRNA expression of myogenic markers (e.g. myosin heavy chain; MHC) and by morphology, fusion index, and dimensions of myotubes after immunostaining and confocal microscopy.
Results and discussion
Photo-polymerization of the ELP matrix resulted in a stable gel stiffness of ~100-300 Pa, which was maintained when heating tissue to 80°C to emulate cooking. Vitamin-mediated crosslinking allowed 3D encapsulation of viable cells. Multimaterial printing of ELP fibers with edible gellan gum microgels led to a reinforced stiffness of >104 Pa, in the range of bovine muscle tissue (decellularized: 103 Pa). 3D bioprinting of encapsulated cells in viscosity-adjusted ELP bioinks resulted in high post-printing viability (>90%) and allowed parallel alignment which is reminiscent to the spatial alignment in muscle fibers. Following 7 days of differentiation, myogenic progenitors produced multinucleated MHC-expressing myotubes. This contributed to the overall tissue stiffness and further compacted the protein-based cultured tissue.
Conclusions
The recombinant protein bioinks we developed by 3D bioprinting supported myogenesis and provided texture to cultured muscle-like tissue with applications to cellular agriculture and beyond.
References
[1] Suhar RA et al., Biomacromolecules 2023, 24(12).
[2] LeSavage BL et al., J Vis Exp 2023, 135.
[3] Lee YB et al., Materials 2023, 16(3).
Acknowledgements
The authors would like to acknowledge funding from the Shriram Synthetic Biology Starting Grant, Stanford University.
53381518786
