The rising global demand for meat necessitates the development of sustainable alternatives. Cultured meat, produced via tissue culture, offers a promising solution by reducing reliance on conventional livestock farming, which poses ethical and environmental concerns. While 3D culture methods have enabled the creation of cultured minced meat from bovine myocytes, producing large, steak-like tissues with aligned, mature myotubes remains challenging. Existing methods using porous scaffolds yield isotropic myotube distribution, low density, and lack of contractility. This study aimed to develop a method to produce large, 3D bovine muscle tissue with unidirectionally aligned, contractile myotubes, mimicking the characteristics of real steak meat. This involved optimizing hydrogel composition and electrical stimulation protocols, and developing a method for assembling millimeter-thick tissues from myoblast-laden hydrogel modules.

Previous research in cultured meat production has focused on creating smaller, minced meat-like products using various 3D culture techniques. Methods like culturing myocytes around agarose columns or between anchors have yielded ring-shaped or fiber-shaped tissues, respectively, but these are too small for steak applications. Other approaches utilized porous gelatin or soy protein scaffolds to create larger, block-shaped tissue, but these resulted in poor myotube alignment, low density, and a lack of contractility. This highlights the need for improved methods to create larger, more structured, and functionally similar cultured meat products.

Bovine myocytes were isolated from commercial fresh beef, with flow cytometry confirming >85% myocyte purity. A culture device with anchors and pillars, fabricated using stereolithography, was used to guide muscle tissue formation. Bovine myocytes were embedded in either collagen or a fibrin-matrigel hydrogel and cultured for 14 days. Electrical stimulation (1 Hz, 2 ms duration, 3 V/mm) for 2 hours daily from day 3 onwards was tested. Millimeter-thick tissues were constructed by assembling myoblast-laden collagen modules with striped structures, designed to promote myotube alignment, using a PDMS stamp. The modules were stacked and cultured for 7 days. Immunostaining (α-actinin) and confocal microscopy were used to analyze myotube alignment and maturation. The breaking force of the cultured tissue was compared with commercially available beef tenderloin to assess texture. Microbial contamination was determined using a general viable bacteria test.

The study found that a fibrin-matrigel hydrogel combined with electrical stimulation resulted in the highest contractility (100% of tissues) compared to collagen alone (33% contractility). Electrical stimulation improved myotube occupancy (20% to 31%) and the proportion of mature myotubes with α-actinin striped patterns (50% in fibrin-matrigel with stimulation). Myotubes were consistently aligned along the long axis of the tissue irrespective of the hydrogel used or electrical stimulation. The assembly of myocyte-laden modules successfully created millimeter-thick (8 mm × 10 mm × 7 mm) tissues with uniformly distributed, aligned myotubes. The breaking force of the 14-day cultured tissue approached that of beef tenderloin after heating, indicating an increase in stiffness with longer culture. Importantly, microbial contamination in the 14-day cultured tissue was below the detection limit (<5 cfu/g), significantly lower than in commercial beef tenderloin (1.7 × 10⁵ cfu/g).

This study successfully addressed the challenge of creating large, contractile bovine muscle tissue suitable for cultured steak. The optimized culture conditions (fibrin-matrigel and electrical stimulation) resulted in highly aligned, mature myotubes. The modular assembly method allowed for the creation of millimeter-thick tissues with improved texture and significantly lower microbial contamination compared to commercial meat. The findings demonstrate a feasible approach toward producing scalable cultured steak, addressing concerns around sustainability and food safety. The similar response of bovine myocytes to electrical stimulation as observed in other species suggests a conserved mechanism for myotube maturation.

This research presents a significant advancement in cultured meat technology by demonstrating the production of millimeter-thick bovine muscle tissue with desirable structural and functional properties, including contractility and low microbial contamination. The modular assembly approach is scalable, potentially leading to large-scale production of cultured steak. Future work should focus on replacing the inedible hydrogels with edible alternatives, optimizing electrical stimulation parameters, and developing serum-free culture media to enhance the cost-effectiveness and consumer acceptance of this technology.

The study used inedible hydrogels (collagen and fibrin-matrigel). Future research should explore the use of edible alternatives that maintain the beneficial effects on myotube development. The current method involves manual assembly of modules, which limits scalability. Automation, such as 3D bioprinting, could address this limitation. Further investigation into the optimization of electrical stimulation parameters and serum-free culture media is also warranted.