The global demand for meat is projected to increase significantly, raising concerns about animal welfare and environmental sustainability of traditional meat production. Cultivated meat, produced using tissue engineering and animal cells, offers a potential solution. However, current methods often rely on synthetic or animal-derived biopolymers for 3D scaffolds, limiting scalability and raising ethical concerns. This research aims to address these limitations by developing a plant-based scaffold for cultivated meat production. The focus is on using zein, a corn protein, and alginate, a brown algae derivative, to create a biocompatible, biodegradable, and highly stretchable fiber scaffold that promotes muscle cell alignment. The successful development of such a scaffold would contribute significantly to the sustainable and ethical mass production of cultivated meat, offering a more environmentally friendly and humane alternative to conventional meat production methods. The use of plant-based materials is key to overcoming limitations of current methods which often use animal derived or synthetic materials, and thus the use of zein and alginate directly addresses this crucial point of sustainability. The ability to create aligned muscle tissues is important for replicating the texture and properties of real meat and this is an area where this research contributes new findings.

Numerous studies have explored the use of various biomaterials for 3D scaffolds in cultivated meat production. Synthetic and animal-derived polymers have been widely used, but plant-based alternatives are gaining attention due to their sustainability, biocompatibility, and biodegradability. Soy protein, wheat gluten, and modified alginate have shown promise in supporting muscle cell growth. Zein, a corn protein, is particularly appealing due to its biocompatibility, biodegradability, and FDA approval as a food ingredient. However, pure zein's poor rheological and hydrophobic properties hinder its direct application in scaffold fabrication. Existing muscle cell alignment methods often struggle with scalability, posing a challenge for mass production of cultivated meat. This review of the literature highlights the need for a sustainable and scalable method of creating a biocompatible scaffold that can be used to create aligned muscle fibers that can be used in the mass production of cultivated meat. Existing methods have limitations in achieving this goal.

Zein-coated alginate fibers (ZA fibers) were fabricated using a wet-spinning technique. Alginate solution was injected into a coagulation bath containing a zein solution and calcium chloride (CaCl2). The hydrophobic zein coated the hydrophilic alginate fiber, forming a zein shell. The optimal zein concentration (30%) and fiber diameter were determined by evaluating the effects on mechanical properties and cell behavior. Scanning electron microscopy (SEM) was used to characterize the fiber structure. Cell viability and adhesion were assessed using a live/dead assay with C2C12 cells (a mouse myoblast cell line) and primary bovine satellite cells (BSCs). A 3D-printed stretching device was used to apply controlled strain to bundles of ZA fibers seeded with cells to promote muscle cell alignment. Immunofluorescence staining with α-tubulin and desmin was employed to assess cell alignment and myotube formation. The expression levels of myogenic genes (Pax7, MyoD, MyoG, MYH1) were analyzed using RT-qPCR. For cultivated meat production, BSCs, bovine preadipocytes, and a bovine endothelial cell line (CPAE) were cultured on ZA fibers to generate muscle, fat, and vessel fibers, respectively. These fibers were then assembled, and the resulting cultivated meat was pan-fried to assess texture and structural integrity. The mechanical properties of the ZA fibers were determined by tensile testing. The methodology section details the exact procedures used to optimize the fabrication of the zein-coated alginate fibers, the characterization of the fibers, the seeding and culture of cells, the application of strain to align muscle fibers, and the assembly of muscle, fat and vessel fibers to produce the artificial meat.

The study successfully optimized the fabrication of ZA fibers, achieving a stable zein coating at a 30% zein concentration. Smaller fiber diameters, obtained by using smaller needles, resulted in better cell alignment. ZA fibers exhibited significantly higher tensile stress, elastic modulus, and strain at break compared to alginate fibers alone, especially after soaking. The zein coating dramatically improved cell viability and adhesion, preventing cell clumping. Applying physical stretch stimulation (up to 75% strain) to bundles of ZA fibers led to highly aligned muscle cells and increased myotube length and desmin expression. The expression levels of myogenic genes (MyoD, MyoG, MYH1) increased significantly with higher strain. The assembly of muscle, fat, and vessel fibers resulted in a cultivated meat product that retained its structure after pan-frying, exhibiting a fibrous texture similar to real meat. The key findings of this paper show the successful production of a plant based biocompatible scaffold that can be used to create cultured meat that closely resembles the texture and properties of animal meat. These results directly address the need for more sustainable and ethically responsible methods of producing meat.

The findings of this study demonstrate the feasibility of using plant-based materials to create a biocompatible and highly stretchable scaffold for cultivating aligned muscle tissue. The use of zein enhances the cell-adhesive properties of alginate, leading to improved cell viability and proliferation. The ability to align muscle cells through physical stretching offers a simple, scalable, and cost-effective approach compared to other techniques, particularly for mass production of cultivated meat. The successful assembly of muscle, fat, and vessel fibers further validates the potential of this method to create a product that closely resembles real meat in terms of both composition and texture. This research contributes significantly to the development of sustainable and ethical cultivated meat production, offering a viable alternative to traditional animal agriculture. The research shows the potential for this approach to be used to scale up the production of cultured meat and make it more affordable for consumers.

This research successfully developed a cost-effective, plant-based fiber scaffold for cultivating and aligning muscle cells, paving the way for sustainable cultivated meat production. The use of zein and alginate, coupled with a simple physical stretching method, addresses major challenges in scalability and ethical concerns associated with existing methods. Future research should focus on mitigating zein's characteristic aroma and flavor to enhance consumer acceptance and explore the scalability and cost-effectiveness of this approach for large-scale production. Further studies may consider optimizing the fiber composition to enhance the sensory properties of the cultivated meat and investigating the long-term stability and shelf life of the product.

The study primarily utilized C2C12 cells and bovine cells; further investigation is needed to confirm the findings with other cell types. The sensory properties of the cultivated meat (specifically, the taste and aroma potentially impacted by zein) require further optimization. The long-term stability of the scaffold and the potential for large-scale production also need to be investigated more thoroughly. Additional research is needed to ensure the consistent quality and performance of plant-derived materials to maintain production stability and consistency.