Cultured meat production is gaining traction due to ethical, economic, environmental, and public health concerns. While plant-based meat analogs are commercially available, the demand for cultured meat mimicking the texture and flavor of real meat remains high. Current cultured meat lacks the structured, fiber-aligned composition of natural steak. This study focuses on creating a structured, steak-like cultured meat using bovine cells. Bovine cells can be obtained either by isolating specific cell types (muscle satellite cells, adult stem cells, multipotent stem cells) from edible muscle tissue or by differentiating induced pluripotent stem cells (iPSCs). While primary cells, such as muscle satellite cells, maintain differentiation potential for a limited number of passages, they are considered safe for consumption. Previous attempts at creating cultured steak have faced challenges in replicating the aligned muscle fibers and interspersed fat and blood vessels characteristic of real steak. 3D bioprinting, especially supporting bath-assisted 3D printing (SBP), offers advantages in scalability and structural control for tissue engineering. SBP overcomes limitations in ink viscosity and drying issues encountered in air-interfaced extrusion-based 3D printing. Steak meat possesses a unique aligned structure of skeletal muscle fascicles connected by tendons, allowing for contraction and relaxation. The ratio and location of muscle, adipose, and blood capillaries vary across different meat cuts and origins. This research aims to develop a method for assembling these three cell types with precise control over location, ratio, and quantity to create engineered cultured steak.

The existing literature highlights the challenges in creating structured cultured meat, particularly steak. Several tissue engineering techniques like cell sheet engineering, cell fiber engineering, 3D printing with scaffolds, and 3D cell printing have been explored. 3D cell printing shows promise for its scalability and control over structure and composition. Supporting bath-assisted 3D bioprinting (SBP) is a noteworthy technique as it addresses issues of ink viscosity and drying during printing. Studies have demonstrated the feasibility of SBP in fabricating complex tissues. The aligned structure of steak, with its muscle fascicles connected to tendons, requires precise control over cell fiber arrangement. Different meat types exhibit varying ratios of muscle, adipose, and blood vessels, influencing the overall texture and flavor. The need for a method to assemble these cell types accurately is crucial for creating realistic cultured steak.

This study employed a three-step strategy for creating cultured steak: 1) isolation and expansion of bovine satellite cells (bSCs) and bovine adipose-derived stem cells (bADSCs); 2) development of tendon-gel integrated bioprinting (TIP) for fabricating cell fibers and their differentiation into skeletal muscle, adipose, and blood capillary fibers; 3) assembly of the differentiated fibers to create engineered steak-like tissue. bSCs were isolated from masseter muscle of Japanese Black cattle using collagenase treatment and fluorescence-activated cell sorting (FACS), selecting CD31-, CD45-, CD56+, CD29+ cells (Pax7+ bSCs ~80%). Proliferation and differentiation potential were assessed using 2D culture with a p38 inhibitor to maintain differentiation capacity up to passage 8. Adipogenic differentiation of bADSCs was evaluated in 3D culture with collagen microfibers (CMF)/fibrin gel, optimizing the medium with a mixture of seven free fatty acids and a TGF type I receptor ALK5 inhibitor to enhance lipogenesis. Endothelial differentiation of bADSCs was investigated using horse serum (HS) as an inducer, confirmed by CD31 expression and tubulogenesis on Matrigel. SBP was initially used for muscle fiber fabrication, printing bSCs with fibrinogen and Matrigel in a supporting bath of gelatin or gellan gum. Needle anchoring was used to maintain fiber structure during contraction. TIP was then developed by integrating tendon gels into the SBP process, anchoring printed cell fibers to resist contraction and maintain structure. Muscle, fat, and vascular cell fibers were fabricated individually using TIP, with optimization of media conditions for each cell type. The DNA amount, compressive modulus, and water content of TIP-derived cell fibers were compared to commercial beef. Finally, muscle, fat, and vascular fibers were manually assembled, treated with transglutaminase, and shaped into a steak-like structure mimicking the histological structure of Wagyu beef.

The study successfully isolated and characterized bovine satellite cells (bSCs) and bovine adipose-derived stem cells (bADSCs), verifying their proliferation and differentiation potentials. Optimal conditions for adipogenesis in bADSCs were established using a combination of seven free fatty acids and an ALK5 inhibitor, resulting in significant upregulation of PPARγ2 and FABP4, key adipogenic markers. Horse serum was identified as a potent inducer of endothelial differentiation in bADSCs. The tendon-gel integrated bioprinting (TIP) method was successfully developed and shown to effectively create aligned muscle fibers by resisting cell contraction. TIP resulted in the fabrication of muscle, fat, and vascular cell fibers with characteristics comparable to those in commercial beef, although some differences in DNA concentration and water content were observed. The assembled engineered steak-like tissue, constructed by manually assembling the individually-produced muscle, fat, and vascular fibers, successfully replicated the histological structure of commercial Wagyu beef.

This research presents a novel approach for constructing whole-cut meat-like tissue composed of edible bovine cells. The detailed characterization of bovine stem cell behavior and differentiation, particularly the adipogenic and endothelial differentiation of bADSCs, is a significant contribution. The development of TIP addresses a critical challenge in cultured meat production—maintaining fiber alignment and structure during muscle cell differentiation. The successful creation of a small-scale, structured cultured steak demonstrates the potential of TIP for producing realistic, whole-cut cultured meat. However, the study's limitations, such as the small size and inedibility of the current prototype, highlight the need for further research. Future work should focus on scaling up the TIP process, developing edible culture media and bioprinting materials, and optimizing the process to achieve desired tenderness, flavor, and nutritional components.

This study successfully demonstrated the fabrication of a small-scale, structured cultured steak using a novel tendon-gel integrated bioprinting (TIP) method. The detailed characterization of bovine cell behavior and the development of TIP represent significant advancements in cultured meat technology. Future research should focus on scaling up the production process, ensuring edibility, and optimizing the composition to achieve the desired taste and texture of commercial beef.

The current study produced a small, proof-of-concept steak-like structure that is not yet ready for consumption. Scaling up the TIP method for large-scale production will require further optimization. The manual assembly process is not suitable for industrial applications. The edibility of all materials used in the process needs to be further confirmed. Future research needs to address the texture, flavor, and nutritional profile of the cultured meat to meet consumer expectations.