Background <p>Mesenchymal stromal cells (MSCs) are currently employed in numerous clinical protocols, and have been used to enhance the regeneration of bone tissue in case of osteonecrosis, long bone and maxillary bone defects. Traditionally, these protocols were designed using freshly harvested, cultured MSCs. However, several limitations arose due to logistical issues, revealing the need to modify the protocol to facilitate the clinical process. In this context, cryopreservation and the use of the MSCs immediately after thawing seem to be the easiest way to overcome most of these constraints.</p> Methods <p>Bone marrow MSCs (BMSCs) of three donors were compared immediately after harvesting or thawing. First, the kinetics of cell viability and the gene expression profile were assessed in vitro. Then, the role of the cells in supporting vasculogenesis and bone formation in vivo was assessed using molecular biology and histology.</p> Results <p>Firstly, we observed a reduction in cell viability immediately after thawing, but no difference after 2&#xa0;h. Furthermore, the gene expression profile was equivalent for genes involved in osteoblastic differentiation, vasculogenesis, inflammatory cytokines and proliferation. These in vitro results were confirmed by in vivo assays, which showed that cryopreservation did not affect their inflammatory response or vasculogenesis potential. Additionally, analysis of cell survival kinetics and bone formation assays revealed that cryo- and fresh-BMSCs exhibit equivalent potential to induce new bone formation and participate directly in bone formation, as evidenced by the expression of human osteoblastic genes.</p> Conclusion <p>Our study demonstrated that cryo-BMSCs possess the same properties as fresh BMSCs. Therefore, using cryo-BMSCs appears to be the optimal approach for future bone tissue engineering protocols and will facilitate the establishment of a BMSCs bank for future clinical trials.</p>