Taken together, these findings demon

Taken together, these findings demonstrate that coculture spheroids may be beneficial for various regenerative medicine applications. However, their fabrication and characterization is also associated with many challenges. In fact, the viability and function of individual cell types in such coculture spheroids is crucially dependent on the used cell ratios during spheroid generation [46]. Accordingly, identifying the ideal cell ratio is a first mandatory prerequisite for the successful use of coculture spheroids in tissue engineering. In addition, the cell distribu- tion patterns underlie highly dynamic changes over time in coculture spheroids [51]. Therefore, their regenerative capacity may not be constant but, instead, determined by their developmental stage. Another problem is the differentiation of specific biological responses of different cell types in coculture spheroids to external stimuli. For this purpose, sophisticated imaging technologies, which are able to penetrate tissue depths and repeatedly analyze distinct cell populations without destroying the spheroid structure, are required [25]. Finally, defining ideal culture conditions for the generation of coculture spheroids is difficult because different cell types may need specific media and supplements to guarantee their long-term survival and function. To overcome this obstacle, monoculture spheroids of different cellular origin or differentiation stage may alternatively be combined in larger tissue constructs. For instance, adipose-derived MSC spheroids osteogenically differentiate in vitro to building blocks for bone tissue engineering, which, however, markedly impairs their in vivo vascularization capacity [51]. Therefore, it may be reasonable to transfer adipose-derived MSC spheroids of varying differentiation stages into bony defects to simultaneously promote vascularization and bone formation. A similar approach has already been described by Alajati et al. [52]. They coimplanted osteoblast spheroids and human umbilical vein endothelial cell (HUVEC) spheroids in subcutaneous pockets of immu- noincompetent mice. This approach resulted in the formation of grafted human endothelial cell- derived, host mural cell-covered blood vessels, which were interspersed among the outgrowing bone matrix-producing osteoblasts.