Patient-derived ependymoma and medulloblastoma tumoroids: generation, biobanking and drug screening.

Ependymoma and medulloblastoma are among the most common malignant pediatric brain tumors and contribute significantly to morbidity and mortality in affected children. Robust models for investigating these tumors' biology and heterogeneity, and exploring alternative therapeutic options, are currently limited. Here we present a detailed protocol for the generation and maintenance of pediatric patient-derived tumoroids (pPDTs) and pediatric patient-derived xenograft tumoroids (pPDXTs) directly from primary ependymoma and medulloblastoma tumor specimens. The protocol extension expands on our previous method for human induced pluripotent stem cell-derived medulloblastoma and high-grade glioma cancer organoids, with which it shares key reagents and methodological steps. This optimized workflow ensures efficient tumoroid establishment, amplification, biobanking, cryopreservation and recovery. In addition, we describe a scalable, low-throughput drug screening approach using calcein-based live-cell staining and automated image analysis, enabling rapid assessment of therapeutic responses. This protocol provides a robust and reproducible platform for modeling pediatric brain tumors in vitro and will enable broader adoption of patient-derived tumoroid systems for mechanistic studies and preclinical drug screening in pediatric neuro-oncology research. The protocol takes 28-35 days for the generation of tumoroids and from 1 to 4 weeks for amplification, biobanking and downstream applications. The protocol requires at least 3-6 months to become proficient in handling patient-derived samples and generating tumoroids.