The global meat demand is rising, but conventional meat production significantly contributes to greenhouse gas emissions. Cell-based meat offers a sustainable alternative, reducing environmental impact. Two main methods for creating 3D tissue exist: scaffold-based and scaffold-free. Scaffold-based methods often use animal-derived materials, necessitating the development of more sustainable techniques. Cell sheet technology, using temperature-responsive culture dishes (TRCDs), provides a scaffold-free approach. TRCDs utilize poly(N-isopropylacrylamide) (PIPAAm), allowing cells to adhere at 37°C and detach at lower temperatures, forming cell sheets that can be stacked to create 3D tissue. This technology has been successfully used for various tissue engineering applications. This study aimed to create scaffold-free cell-based meat using this technology and characterize its texture and nutrients.

Existing literature highlights the environmental concerns associated with traditional meat production and the potential of cell-based meat as a solution. Studies have explored both scaffold-based and scaffold-free methods for 3D tissue construction. Scaffold-based methods often utilize animal-derived materials, a limitation addressed by the scaffold-free approach of cell sheet technology. Previous research demonstrates the successful application of cell sheet technology in creating various functional tissues, including skeletal muscle, liver tissue, and cardiac tissue, underscoring its potential for cell-based meat production.

Bovine myoblast cells were isolated from bovine cheek muscles and cultured in TRCDs. Cell sheets were harvested by lowering the temperature to 20°C. Ten cell sheets were stacked to form 3D tissue constructs. The dimensions (diameter and thickness) of the cell sheets were measured using ImageJ software and optical coherence tomography. The cytoskeleton of the cells was visualized using F-actin staining. Histological analysis (HE and AZAN staining) and immunostaining (PAX7 and MYOD) were performed to assess cell structure and composition. Texture profile analysis (TPA) was conducted on both raw and boiled cell sheet-based meat to evaluate texture parameters (hardness, elasticity, cohesiveness, springiness, chewiness, brittleness, adhesiveness). Nutritional analysis included measurements of water content, total protein (Bradford method), carbohydrates (phenol-sulfuric acid method), and unsaturated fatty acids (vanillin-sulfate method). Large-sized cell sheet-based meat was produced using larger TRCDs. The reusability of TRCDs was also evaluated.

The diameter of the bovine myoblast cell sheets decreased, and thickness increased with longer culture durations. The volume of the 10-layered constructs remained relatively constant. F-actin staining revealed increased F-actin with longer culture times. HE and AZAN staining showed no significant structural differences, and no collagen fibers were observed. TPA showed that hardness increased with culture duration and further increased after boiling, mirroring the behavior of natural meat. Compared to beef, the cell sheet-based meat had a significantly lower wet weight percentage of protein (5-6% vs 11.3%) but a higher dry weight percentage (42.3-50.6% vs 28.8%). The carbohydrate content was significantly higher in the cell sheet-based meat than in beef, likely due to the high-glucose culture medium. The unsaturated fatty acid content was lower in the cell sheet-based meat than in beef, with high variability in the beef samples. The method was successfully scaled up to produce large-sized cell sheet-based meat constructs.

This study successfully demonstrated the production of scaffold-free cell-based meat using cell sheet technology. The observed increase in hardness with culture time and after boiling suggests that actin filament expression plays a crucial role in texture development. The difference in protein content between wet and dry weight comparisons highlights the high water content of the cell sheet-based meat. The higher carbohydrate content can potentially be reduced by using a lower glucose culture medium. The scalability of the method is a significant advantage, paving the way for large-scale production. The use of animal-derived materials is minimal, aligning with the principles of sustainable food production. Further research could explore strategies to optimize the texture and nutritional profile, such as promoting myotube formation and investigating alternative culture media.

This research demonstrates the feasibility of producing scaffold-free cell-based meat using cell sheet technology. The method offers scalability and minimizes animal-derived components. Future work should focus on enhancing the texture and nutritional composition, possibly through manipulation of myoblast orientation, myotube formation, and culture conditions. Investigating the use of non-plastic materials for TRCDs would also enhance sustainability.

The study focused on bovine myoblast cells and may not be directly generalizable to other cell types. The protein content of the cell sheet-based meat is lower than beef by wet weight, although this is due to higher water content. The high carbohydrate content might be considered a limitation from a nutritional standpoint. More extensive nutritional analysis beyond the parameters measured would be beneficial.