Cellular agriculture is a promising avenue for sustainably contributing to the growing food demand. In this respect, cultured or in vitro meat receives increasing attention. Yet, there is currently no scientific evidence that animal myofibers equivalent to meat as a nutrient dense, highly structured food, can be made in vitro. This proposal will address several existing hurdles. A major challenge is to obtain a suitable cell type capable of both sufficient proliferation and robust differentiation. Muscle adult progenitor cells have a very good differentiation capacity, yet weak proliferation capacity, whereas mesenchymal and embryonic stem cells display the opposite behavior. First, we will compare and enhance the proliferation and differentiation capacity of these three cell types, resulting in an informed choice of cell type for myofiber generation. Secondly, we will design a ‘smart matrix’, including food-grade components that contribute to texture, flavor and a good nutritional profile. This involves a proper choice of materials which is mutually dependent on the technological approaches to create a 3D structure. Physicochemical stability of, and cellular adhesion and survival on these new matrices will be investigated. The best performing smart matrices will be tested on their potential to support muscle cell differentiation. These insights will be subsequently used to increase muscle fiber size and to study in vitro muscle development in a 3D architecture. In addition, strategies for stimulating the build-up of contractile proteins will be investigated. Finally, public acceptance, consumer attitudes, perceptions and intentions, as well as impact on sustainability will be assessed. This project will deliver a sound scientific basis and assessment of key technologies for advancing knowledge in this field and allowing informed decision making for downstream valorization strategies.