Cultured meat, a sustainable alternative to conventional meat production, is gaining significant traction. However, current research faces challenges in replicating the complex biological and physical properties of animal-derived meat, particularly its organoleptic characteristics (sensory properties). This study addresses this gap by focusing on the control of cell differentiation to optimize the sensory qualities of cultured meat. The quality of meat is largely determined by the characteristics of its muscle and fat tissues, including myofiber dimensions and lipid content, which are in turn influenced by cellular differentiation. The researchers hypothesize that by manipulating the mechanical properties of the scaffold, they can control the differentiation of muscle and fat cells, ultimately producing cultured meat with enhanced organoleptic properties similar to conventional beef. Previous research has explored various scaffolds for cultured meat production, including 3D-printed bioinks, plant-based materials, and soy proteins. However, the impact of scaffold stiffness on the nuanced food characteristics of the final product has largely been overlooked. This study aims to bridge this knowledge gap by meticulously controlling scaffold stiffness to regulate myogenesis (muscle cell differentiation) and adipogenesis (fat cell differentiation) and subsequently evaluating the effect on the sensory properties of the cultured meat. The overall objective is to develop a method of producing cultured meat that closely matches the sensory experience of consuming animal-derived meat, thereby increasing its market appeal and viability as a substitute.

Existing literature highlights the importance of muscle fiber properties and lipid content in determining meat quality and sensory attributes. Studies have demonstrated correlations between myofiber dimensions and tenderness, as well as the relationship between lipid content and flavor. Research on scaffold engineering for tissue engineering has shown that scaffold stiffness can influence cellular functions, including differentiation rates. However, the application of this principle to the production of cultured meat with tailored organoleptic properties remains relatively underexplored. While various scaffolds have been investigated for cultured meat production, the focus has largely been on cell proliferation and the macroscopic structure of the meat, often neglecting the intricate relationship between scaffold properties, cellular differentiation, and the resulting sensory characteristics. This study builds upon this existing body of research by integrating scaffold engineering with controlled cell differentiation to precisely manipulate the quality of cultured meat.

The researchers fabricated 2D hydrogel scaffolds composed of fish gelatin and alginate. The stiffness of the hydrogels was precisely controlled by varying the concentration and crosslinking degree of alginate. Two alginate concentrations (low and high) and two crosslinking degrees (low and high) were tested, resulting in four distinct hydrogel types. Raman spectroscopy was employed to verify the successful manipulation of the alginate crosslinking degree. Compression tests determined the stiffness of each hydrogel, selecting those with stiffnesses mimicking those of adipose tissue (3-4.5 kPa) and skeletal muscle tissue (10-12 kPa) for further experiments. Bovine primary myoblasts and adipose-derived mesenchymal stem cells (adMSCs) were isolated from animal tissues. The weight loss of the selected hydrogels was measured in the presence of the myoblast-conditioned medium to evaluate their stability under cell culture conditions. Myoblasts were cultured on the chosen hydrogels, and their differentiation was monitored using confocal microscopy (for MHC expression) and proteomic analysis (LC-MS/MS). The amount of myosin heavy chains (MHCs) was quantified using ELISA. The total protein content was also assessed using a BCA assay. The texture of the muscle constructs was evaluated via a rheometer before and after grilling. A gas chromatography (GC)-MS analysis identified the volatile compounds responsible for the flavors of the grilled samples. Similarly, adMSCs were cultured on the selected hydrogels, and their adipogenic differentiation was assessed using Oil Red O staining and confocal microscopy (LipidTOX staining). Proteomic analysis (LC-MS/MS) was conducted, and the fatty acid flavor profile of the constructs was analyzed via GC-MS after grilling. Finally, muscle and fat blocks were assembled to create a small-sized cultured beef using microbial transglutaminase (mTG) as a crosslinker, replicating a 3:1 muscle-to-fat ratio found in beef brisket. The nutritional content, texture (texture profile analysis, TPA), and flavor profile of the assembled cultured meat (ACM) were then compared to those of actual beef. The process was extended to build an engineered T-bone steak by assembling muscle and fat blocks with varied muscle:fat ratios, emulating a strip loin and tenderloin. The resultant T-bone steak was also grilled and analyzed.

The stiffness of the gelatin/alginate hydrogel scaffolds significantly impacted the differentiation of both myoblasts and adMSCs. Hydrogels with higher stiffness (~11 kPa) promoted myogenesis, resulting in increased MHC expression, higher protein content in the muscle constructs, and enhanced texture and flavor profiles after grilling. The higher stiffness triggered significant upregulation of genes related to muscle proteins and cytoskeleton components. GC-MS analysis revealed a significant increase in Maillard reaction products, including benzaldehyde and 2,5-dimethylpyrazine, known for their savory and roasted beef-like flavors. On the other hand, softer hydrogels (~3 kPa) enhanced adipogenesis, increasing lipid droplet formation in adMSCs and leading to a greater abundance of fatty acid flavor molecules (nonanal and 2-ethyl-1-hexanol) after grilling. The assembled cultured meat (ACM), fabricated by combining muscle and fat blocks with different stiffness, displayed similar nutritional composition, texture (cohesiveness, springiness), and flavor profiles to conventional beef brisket. The engineered T-bone steak, constructed by assembling muscle and fat blocks with different ratios, also successfully demonstrated the potential to produce cultured meat that mimics the structural and sensorial aspects of various beef cuts.

This study's findings directly address the research question by demonstrating the critical role of scaffold stiffness in controlling cellular differentiation and, subsequently, the organoleptic properties of cultured meat. The successful replication of beef-like sensory characteristics in the ACM and engineered T-bone steak highlights the potential of this approach to address a significant limitation in cultured meat production. The results show the profound effect that precise control over the microenvironment can have on the final product's quality. The ability to independently control myogenesis and adipogenesis, leading to an accurate replication of muscle and fat ratios, is a significant advancement in the field. This method offers flexibility and control previously unavailable in cultured meat production. The use of 2D hydrogels simplifies the process, eliminating complexities associated with 3D bioprinting and improving scalability. These findings have broad implications for the future of cultured meat production, paving the way for a more sustainable and efficient method to produce meat products with enhanced sensory appeal. Further research could explore the application of this method to other types of meat, and the optimization of adipocyte maturation within the scaffolds could lead to even greater improvements in fat quality.

This study demonstrates a novel approach to producing cultured meat with enhanced sensory and nutritional properties by precisely controlling cell differentiation via scaffold engineering. The ability to independently control myogenesis and adipogenesis through scaffold stiffness manipulation allows for the creation of meat constructs with desired muscle-to-fat ratios and enhanced organoleptic qualities. The successful replication of conventional beef characteristics in the ACM and engineered T-bone steak demonstrates the potential for widespread adoption of this method in the cultured meat industry. Future research could focus on optimizing the scaffold materials, exploring different cell types, and scaling up the production process for commercial applications. Moreover, investigations into further refining the control of cell differentiation and adipocyte maturation are warranted.

The study focused on a specific type of beef cut (brisket and T-bone steak) and utilized bovine cells. The generalizability of these findings to other types of meat and animal species requires further investigation. The maturity of adipocytes was not fully evaluated in this study, warranting future research in that area. While the flavor profiles were similar, some minor differences in specific volatile compounds were observed between the ACM and conventional beef, indicating room for optimization.