Fetal bovine serum (FBS) is a widely used supplement in cell culture media which provides cells with vital factors including growth factors, hormones and vitamins essential for cell survival, growth and division. However, the use of FBS in in vitro cell culture is controversial. FBS is a variable and undefined medium supplement with unknown and complex composition. It may contain unpredictable factors that have an influence on cell responses which can even change between batches. FBS-containing medium should be avoided wherever possible. Alternatively, a number of chemically defined animal-component-free media are commercially available, which are specific for different cell types. However, their exact formulations are not released to customers. This makes it difficult to determine the effect of soluble factors on cell behavior. In bone tissue engineering, a 3D construct should be able to promote mesenchymal stromal cells (MSCs) to differentiate towards the osteogenic lineage, to produce collagen and to deposit a mineralized extracellular matrix. Glucose is one of the essential components in cell culture medium which provides cells with energy for metabolic processes such as proliferation and matrix production. It has been shown previously that glucose concentration in the medium has an effect on osteogenic differentiation and also depends on the type of FBS used. In this study we aim for two goals: i) to develop a fully defined serum substitute medium and ii) to compare the effect of different glucose concentrations in that serum substitute medium on the osteogenic differentiation of MSCs in vitro.