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Osteosarcoma (OS) is the most common primary bone malignancy with high rates of recurrence and metastasis. OS often spreads to lungs, an optimized model for studying lung metastatic OS cells may help develop potential therapies for patients with lung metastasis. Here we firstly report an organoid culture system for lung metastatic OS tissues. We provided a fully described formula that was required for establishing lung metastatic OS organoids (OSOs). Using this protocol, the lung OSOs were able to be maintained and serially propagated for