Expansion of mesenchymal stromal cells (MSC) is one of the key steps for their use in tissue engineering or cell therapies. To increase cells expansion yields, two milestones have to be achieved that will allow a wider MSC therapeutic use, namely an optimal serum-free medium and a process intensification via 3D suspension culture on microcarriers . Indeed, one of the major obstacles to obtain a reliable manufacturing process is that most of MSC cultivation methods still rely on media being supplemented by a significant volume of fetal calf serum. While efforts have been made to develop serum-free media (SFM) for MSC expansion, they were systematically designed for planar plastic cultivation systems. The aim of this study was to compare usual serum alternatives on 2D and 3D cultures. However, these alternatives can be ill-defined, either when the medium formulation is proprietary or when it has been humanized with blood derivatives. Thus several supplements were also investigated to design a more defined serum-free formulation that could be used to expand stem cells on microcarriers.