A 3D Bone Culture Platform Using Human Osteocytes and Decellularized Extracellular Matrix for Modeling Musculoskeletal Diseases.

Modeling musculoskeletal diseases such as osteoporosis requires in vitro platforms that accurately reproduce key features of human bone biology. Conventional 3D culture systems provide valuable insight into cell-cell and cell-matrix interactions, but their dependence on costly materials, bioprinters, or specialized infrastructure limits their broader adoption. Here, we describe a robust and cost-effective protocol for generating 3D bone constructs using human-derived osteocytes embedded in GelMA hydrogels, supplemented with decellularized extracellular matrix (dECM) obtained from donated femoral heads. This pipetting-based method enables the fabrication of reproducible, bone-like constructs at physiological temperature (37 °C), using standard laboratory equipment. The resulting constructs maintain high viability and structural integrity, supporting downstream applications such as immunofluorescence imaging and molecular assays. The incorporation of patient-derived dECM enhances translational relevance by better reflecting human bone remodeling and disease progression. This approach provides an accessible, scalable platform for drug testing, regenerative medicine, and mechanistic studies of bone biology. Overall, the protocol offers a practical alternative to high-cost bioprinting techniques and can be readily adapted to other tissue-specific dECM sources.