Abstract Unlike many high-grade tumors, low-grade glioma (LGG) has faced challenges in advancing scientific and clinical breakthroughs, affected by the limited capacity to develop representative preclinical tumor models. Major barriers to establishing traditional LGG cell culture systems have been cellular senescence and the need to introduce additional genetic modifications to obtain cultured growth. Such LGG models, while extremely valuable, require extensive periods for development and result in clonally selected lines which may not present the complexity of LGG tumor biology. To mitigate such challenges in LGG model development and address the need for three-dimensional tissue cultures, we have successfully generated organoids from fresh tissue specimens obtained from surgical resection. We have demonstrated success of this method on various pediatric brain tumor histologies and have expanded these approaches successfully to LGG. Through such efforts, we have developed 14 LGG tumors’ organoid models as well as other low-grade tumor’s models such as craniopharyngioma, schwannoma, meningioma, optic glioma, and oligodendroglioma. The tissue was processed immediately post-extraction and cultured in media solution on rotating platform. Organoid growth was observed within 1-3 weeks of initiation and tested for growth for up to 3 months. Phenotypic analysis revealed organoid cell composition representing clinical histology. The immune component was preserved in organoids for up to 40 days. MAPK pathway activity, a hallmark marker for KIAA-BRAF fusion positive pilocytic astrocytoma was preserved for up to 4 weeks. Our successful deployment of three-dimensional patient-tissue derived organoids provides for an efficient, simple workflow for the generation of unique and transformative preclinical models for the LGG field. Supported by the Children’s Brain Tumor network’s open science model, 3D organoids will provide investigations with broader resources for LGG tumor biology preclinical testing and support translational studies on behalf of the development of novel clinical trials.