Development of cell-laden gelAGE microspheres as modules for the bioassembly of larger vascularized hybrid constructs

Abstract

Three dimensional (3D) tissue engineering has shown great potential for the development of in-vitro models and regenerative therapies for which there is a critical need, such as treatment of large bone defects. Yet, limited nutrient diffusion due to the lack of sufficient perfusable vascular networks, and the soft environment needed for vascularization restrict the size, integrity and integration of implantable constructs. The bioassembly of vascularized modules such as microspheres into hybrid construct represents an interesting approach for the creation of larger tissues with heterogeneous mechanical properties and microenvironment. The present study investigated the performance of GelAGE microspheres as a modular system for the development of vascularized 3D cultures that can be assembled into larger perfusable macrotissues, subsequently serving as a potential platform for maturation into vascularized bone. After finding an optimal hydrogel mix formulation, HUVECs and MSCs were encapsulated separately and together as co-cultures into cell-laden GelAGE microspheres, showing high viability one day after fabrication (≈80%), and increasing metabolic activity over 10 days of culture. The microfluidic system used as a scalable automated fabrication method yielded microspheres with minimum size variation and allowed size adjustment. Vascular development throughout 10 days of culture was visually and quantitatively described. Considerable vascular development was seen on the surface of co-culture-laden microspheres. Microsphere fusion capacity and behavior was further characterized after self-assembly in U-well culture plates, and guided assembly on PLA scaffolds. Distinctive fusion behavior was observed for different microsphere combinations, with co-culture-laden microspheres exhibiting the highest fusion capacity forming gapless uniform bodies after merging. Despite missing more data about vascularization below the surface, these first results suggest GelAGE microspheres are a promising system for the de-novo development and assembly of larger vascular networks.