High-throughput microfluidic generation of self-assembled, uniform 3D vascularized and mineralized bone organoids without matrix biomaterials

• High-throughput chip generates 540 uniform, scaffold-free bone organoids for drug screening, disease modeling, and material testing. • hBMSCs, HUVECs, and mineralized collagen self-assemble into organoids within 3 days. • Organoids show high viability, proliferation, and osteogenic/angiogenic potential. Bone organoids hold great promise for modeling bone-related diseases, improving bone injury repair strategies, and enabling high-throughput drug screening. However, conventional approaches rely heavily on matrix biomaterials—such as Matrigel, collagen gels, or 3D-printed scaffolds—which introduce undefined parameters, pose manufacturing complexities, and raise cost barriers, potentially limiting clinical translation. To address these challenges, we present a novel high-throughput microfluidic chip that generates 540 uniformly sized, scaffold-free 3D bone organoids simultaneously within six independent channels. By co-cultivating human bone marrow mesenchymal stem cells (hBMSCs), human umbilical vein endothelial cells (HUVECs), and mineralized collagen (MC), self-assembled bone organoids formed by the third day and progressively compacted over time, exhibiting enhanced cell viability and proliferation. The inclusion of MC upregulated multiple osteogenic markers (OCN, ALP, COL-1, RUNX2, and BMP-2), while endothelial markers (PECAM-1, HIF-1α, and VEGF) remained consistently expressed, reflecting stable vascularization and mineralization potential. Overall, this high-throughput, matrix-free microfluidic platform offers a biomimetic environment for the investigation of osteogenesis and angiogenesis and holds significant promise for advanced material assessment, drug screening, and disease modeling. Schematic of high-throughput microfluidic chip for Multicellular three-dimensional spheroids building in bone regeneration evaluation.