Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. Afterin vitrocharacterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to studyin vivotheir viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry.In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72 h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.