Dairy byproducts as sustainable alternatives to FCS in 2D and 3D skeletal muscle cell cultures.

Skeletal muscle tissue engineering is a rapidly developing field with applications in disease modelling, tissue replacement, biorobotics, and cultivated meat. The need for more sustainable and ethical biotechnologies has grown due to concerns about resource scarcity, climate change, and animal welfare. One major challenge is replacing fetal calf serum (FCS), a widely used but ethically and environmentally highly problematic media supplement. A promising alternative is the utilization of natural byproducts such as whey and colostrum from the dairy industry, which provide essential nutrients and growth factors. In this study, wheys produced by microfiltration of raw milk and colostrum were investigated as FCS replacements for culturing C2C12 skeletal muscle cells. Composition analysis confirmed a variety of pro-proliferative compounds in both substances. Cell culture experiments led to the development of an optimized medium formulation based on colostrum whey. Colostrum whey medium (CM) supported cell proliferation and maintained the myogenic differentiation potential for over 30 days. Additionally, a CM-based freezing solution enabled effective cryopreservation throughout culture. In 3D static suspension culture, CM sustained viable spheroids for over 14 days. Spheroids showed significantly higher proliferation compared to those in serum-containing medium, making CM suitable for 3D modelling and scale-up of biomass production. These findings highlight CM as a sustainable, cost-effective, and ethical alternative for skeletal muscle tissue engineering, particularly in cultivated meat production.