To dates, as stem cells are considered a promising cell source for tissue engineering and regenerative medicine, many culture strategies have been extensively studied to generate in vitro stem cell-based three-dimensional (3D) tissue constructs. Previously, we have reported 3D-osteogenesis of stem cells could be well induced by an integrated 3D-bioprocess composed of hydrogel encapsulation and a fed-batch rotating wall vessel (RWV) bioreactor. However, such a fed-batch based 3D culture system appeared to be limited in a real-time control of nutrients, metabolites, oxygen, and other molecules transport, which hinders to support optimal culture environments for stem cells’ biological behaviors in proliferation, differentiation, and tissue formation. Therefore, in our lab, we designed a novel, automated perfusion RWV bioreactor for continuous supply of nutrients/oxygen and removal of metabolic wastes during the whole culture period to establish optimal culture condition for stem cell-based 3D tissue formation. Our new bioreactor demonstrated highly reproducible ...