Cultured meat offers a sustainable alternative to traditional meat production, addressing critical environmental and ethical issues. A key aspect of this process is the large-scale proliferation of muscle satellite cells, which can further proliferate and form the primary component of cultured meat. However, ensuring efficient cell proliferation within bioreactors is a significant challenge. Without effective and robust cell proliferation, it would be impossible to meet the production demands of cultured meat. Moreover, 3D cell spheroids tend to form tightly packed structures. As these spheroids grow larger, the limited penetration of oxygen and nutrients can lead to the formation of necrotic cores or cause cells in the central layers to experience cell cycle arrest, resulting in either irreversible senescence or reversible quiescence. This adds complexity to maintaining stable cell growth. To address these challenges, different ECM components, specifically Matrigel and Collagen I, were introduced to alter matrix stiffness and growth factor concentrations. The goal was to address issues of reduced cell proliferation and cell cycle arrest. Results demonstrated that in the absence of ECM, 3D-cultured bovine muscle satellite cells spontaneously formed myospheres but exhibited cell cycle arrest and inhibited proliferation. These issues were reversed with the addition of ECM. Increasing ECM stiffness, particularly through higher concentrations of Matrigel and Collagen I, significantly enhanced cell spreading but had a complex effect on cell proliferation. While Matrigel promoted both cell spreading and proliferation, higher stiffness and growth factor levels were associated with reduced proliferation rates, indicating a trade-off between these processes. Notably, a stiffness of 1.5 Pa with 1.56 mg/ml Matrigel yielded the highest proliferation rate, suggesting this condition might be optimal for use in bioreactor systems. Additionally, increasing matrix stiffness using Collagen I also enhanced cell spreading, indicating that cell spreading is strongly influenced by ECM stiffness. Furthermore, Matrigel reduced the expression of quiescence and senescence markers, helping to maintain cells in a proliferative state. These findings underscore the importance of optimizing ECM properties to balance cell proliferation and structural organization in 3D cultures, providing a foundation for scaling up 3D culture systems in bioreactor settings—a critical step toward large-scale cultured meat production. / Thesis / Master of Applied Science (MASc)