Childhood cancer arises from disrupted developmental processes driven by genetic and epigenetic changes, making them fundamentally different from adult malignancies. Understanding this unique biology requires models that faithfully capture the complexity of pediatric cancer. Patient-derived organoids (PDOs) have emerged as a transformative tool in this space, preserving the heterogeneity, plasticity and microenvironmental features of the original tumor in the laboratory setting. This protocol provides research through the practical steps of building PDOs from pediatric tumors, solid tumor biopsies and bone marrow aspirates. These living models are then applied to a range of analytical techniques, including florescence imaging, Western blot analysis, flow cytometry and IHC, to unravel how these tumors develop and behave. Bringing organoid technology into pediatric oncology represents a meaningful step towards accelerating both our biological understanding and the development of better treatments for children with cancer.
