The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.