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Aspiration-assisted bioprinting of co-cultured osteogenic spheroids for bone tissue engineering. Heo Dong Nyoung,Ayan Bugra,Dey Madhuri,Banerjee Dishary,Wee Hwabok,Lewis Gregory S,Ozbolat Ibrahim T Biofabrication Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 d, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling. 10.1088/1758-5090/abc1bf