Cultured meat presents a potential solution to the environmental, ethical, and health concerns associated with traditional animal agriculture. Life cycle analyses suggest that cultured meat could significantly reduce land and water usage, greenhouse gas emissions, and other environmental impacts. However, widespread adoption faces significant challenges, particularly the high cost and reliance on fetal bovine serum (FBS) in current cell culture media. FBS is expensive, unsustainable, and introduces variability, conflicting with the goals of cultured meat production. Existing serum-free media for satellite cell expansion are often complex, ineffective, or contain components raising regulatory concerns. This study addresses this critical limitation by adapting the low-cost B8 serum-free medium, originally developed for pluripotent stem cells, for use with bovine satellite cells (BSCs). The simplicity and low cost of B8 make it an attractive starting point for developing a scalable and sustainable medium for cultured meat production.

The literature extensively documents the challenges of culturing meat-relevant cells, such as BSCs, in serum-free conditions. Many attempts to create serum-free media have resulted in complex formulations, lower growth rates compared to serum-containing media, reliance on proprietary or animal-derived components, or the inclusion of potentially problematic additives like synthetic steroids. No previously reported serum-free medium had demonstrated sustained expansion of satellite cells over multiple passages. The high cost of current media, often dominated by serum or proprietary additives, is a major barrier to commercial viability. The B8 medium, a low-cost serum-free option for human iPSCs, offered a promising alternative due to its simple composition of readily available, potentially food-safe components. This study built upon the success of B8 to create a more suitable medium specifically for BSCs.

The study used primary bovine satellite cells (BSCs) isolated from the semitendinosus muscle of a calf. Cell identity was confirmed through Pax7 and Myosin Heavy Chain (MHC) staining. Short-term growth assays (3 and 4 days) were performed using mixtures of standard growth media (BSC-GM, containing 20% FBS), homemade B8 medium, and commercially available HiDef-B8. Various supplements were tested in B8 to improve cell proliferation, focusing on cost-effectiveness and simplicity. A passaging protocol was developed for long-term culture in the optimized medium (Beefy-9, B8 supplemented with recombinant human albumin). This involved delaying the addition of albumin to allow cell adhesion and the use of a suitable cell adhesion protein. Long-term expansion (seven passages over 28 days) was assessed using different FGF-2 concentrations and compared to BSC-GM and B8 alone. The myogenicity of cells grown in Beefy-9 was confirmed via differentiation assays and immunostaining for myogenic markers. Cost analyses were performed to evaluate the economic feasibility of Beefy-9 and its cost-optimization potential. Further experiments explored the effects of increasing rAlbumin concentration in Beefy-9 on both short and long term cell growth.

Initial short-term assays showed that B8 alone could support BSC growth for three days, but not four. Supplementation with recombinant human albumin (rAlbumin) significantly improved BSC proliferation in B8, resulting in growth comparable to BSC-GM. The addition of rAlbumin led to the creation of Beefy-9, a nine-component medium that outperformed B8 alone. A suitable passaging protocol for Beefy-9 was established, which involved a delay in rAlbumin addition after passaging and using a 1.5 µg/cm² coating of truncated vitronectin (Vtn-N) for enhanced cell adhesion. Long-term growth studies showed that Beefy-9 with either high (40 ng/mL) or low (5 ng/mL) FGF-2 concentrations supported sustained BSC expansion for at least seven passages, while B8 alone ceased proliferation after three passages. The average doubling time in Beefy-9 was approximately 39 hours. Cells cultured in Beefy-9 maintained myogenicity, as evidenced by differentiation assays and expression of myogenic markers. Cost analysis revealed that Beefy-9 was more cost-effective than BSC-GM, particularly when components were purchased in bulk and with reduced FGF-2 concentration. Further optimization by increasing rAlbumin concentration to 3.2 mg/mL (Beefy-9+) resulted in a three-fold improvement in cell number with only a two-fold increase in cost. Lipid accumulation was observed in cells grown in serum-free media, which could be further investigated for its potential impact on cultured meat flavor.

The successful adaptation of B8 for BSCs highlights the potential of simple, cost-effective serum-free media for cultured meat production. The significant impact of rAlbumin on BSC growth suggests its multi-faceted roles in cell culture, stabilizing and delivering essential nutrients while also acting as an antioxidant and potentially protecting against stress. The development of Beefy-9 and Beefy-9+ offers researchers a more affordable and readily accessible alternative to serum-containing media, reducing the financial barriers to cultured meat research. While the current cost-savings are substantial compared to serum-containing media, further optimization is needed to achieve true economic viability for large-scale production. The observed lipid accumulation in serum-free conditions requires further investigation. It may be related to insulin concentrations or point to an undesired adipogenic shift, but it may also offer benefits in terms of meat flavor and texture. This study lays groundwork for future computational approaches to media optimization.

This study successfully developed two serum-free media, Beefy-9 and Beefy-9+, for the sustained expansion of bovine satellite cells, which are cost-effective compared to traditional serum-containing media. The methods used showcase a promising strategy for accelerating cultured meat research and moving towards a more sustainable food system. Further optimization of media composition, particularly focusing on reducing the cost of recombinant proteins, and addressing long-term growth limitations will be critical for achieving industrial-scale cultured meat production. Future studies should explore computational media design, alternative cell-adhesion strategies, and the role of lipid accumulation to further improve both cost and efficiency.

The study focused solely on bovine satellite cells. The optimal composition of Beefy-9 may vary for other cell types or species. The long-term culture observed some lipid accumulation in serum-free conditions, which could affect meat quality. While cost-effective compared to serum-containing media, further cost reductions are needed to reach the scale and affordability required for widespread adoption in commercial settings. The study primarily used a one-factor-at-a-time approach to optimization, which may overlook synergistic effects between media components. Further investigation of using engineered, thermally stable growth factors should be explored.