To establish a sustainable cultured meat technology, a low-cost culture medium must be developed without expensive biological materials such as serum and coating substances. However, even adhering bovine myogenic cells to uncoated culture dishes in the serum-free medium is challenging. We found that serum-free culture medium conditioned by HepG2 and NIH/3T3 cells not only accomplished the cell adhesion on uncoated culture dishes (the serum-containing medium : the serum-free medium : the conditioned medium = 6722 ± 1500 : 2210 ± 319 : 5985 ± 1558 cells/cm2), but also induced proliferation comparable to that observed in a serum-containing medium (the serum-containing medium : the serum-free medium : the conditioned medium = 10,050 ± 2814 : 2200 ± 707 : 8998 ± 3890 cells/cm2). Interestingly, although the nutrient composition of the developed medium differed significantly from that of the serum-containing medium, it tended to coordinate the expression of cell adhesion, proliferation, and myogenic differentiation markers as serum-containing medium. Component analysis and validation experiments suggested that pyridoxamine, asparagine, and glutamic acid contributed to the acquisition of the culture function of the developed medium. Our study paves the way to realize a low-cost and sustainable cultured meat technology.