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BRAIN Publication-derived

Glioblastoma Brain Organoids under Hypoxia

Full SOP with Academic or a plan

Source Hubert et al., 2016 · Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic · 10.1158/0008-5472.can-15-2402

Authors: Christopher G. Hubert, Maricruz Rivera, Lisa C. Spangler, Qiulian Wu, Stephen C. Mack, Briana C. Prager, Marta Couce, Roger E. McLendon, Andrew E. Sloan, Jeremy N. Rich 9 phases Cell source: Patient-Derived Primary Glioblastoma, Xenografts, Genetically Engineered Glioma Models

Abstract

This protocol describes a three-dimensional organoid culture system derived directly from human glioblastoma specimens that recapitulates in vivo tumor architecture, including hypoxic gradients and cancer stem cell heterogeneity. The method supports long-term growth and expansion of tumor organoids from primary patient cultures, xenografts, and genetically engineered models, enabling simultaneous culture of functionally diverse stem and non-stem cell populations within a physiologically relevant microenvironment.

Cell source
Patient-Derived Primary Glioblastoma, Xenografts, Genetically Engineered Glioma Models
Application
Disease modeling; cancer stem cell biology; microenvironmental heterogeneity study

Protocol overview

26 steps across 9 phases

Phase 1 · Tumor Sample Preparation Day 0
  1. a Obtain and Process Glioblastoma Tissue
  2. b Remove Red Blood Cells
  3. c Count Cells and Assess Viability
Phase 2 · Xenograft Dissociation and Magnetic Sorting (if applicable) Day 0-1
  1. a Dissociate Xenograft Tissue
  2. b Magnetic Sorting for CD133
Phase 3 · Pre-Organoid Culture (Tumorsphere Maintenance) Day 0-7
  1. a Culture Cells as Tumorspheres
  2. b Monitor Passage Number
Phase 4 · Organoid Formation and Embedding Day 0-1
  1. a Prepare Matrigel-Cell Suspension
  2. b Form Matrigel Pearls
  3. c Transfer Organoids to Culture Plates
Phase 5 · Organoid Culture and Maintenance Day 1-90+
  1. a Culture Organoids in NBM Complete Media
  2. b Perform Media Changes
  3. c Monitor Organoid Growth
  4. d Document Organoid Growth Timeline
Phase 6 · Organoid Dissociation and Analysis Day 30-365
  1. a Dissociate Organoids for Functional Assays
  2. b Count Viable Cells
  3. c Perform Limiting Dilution Assays
Phase 7 · Orthotopic Xenotransplantation (Optional) Day 150-365
  1. a Prepare Cells for Implantation
  2. b Orthotopic Transplantation into NSG Mice
  3. c Monitor Mouse Health and Tumor Development
Phase 8 · Immunofluorescence and Imaging Day 30-365
  1. a Fix and Prepare Organoid Sections
  2. b Perform Immunofluorescence Staining
  3. c Nuclear Counterstaining
  4. d Image Acquisition
Phase 9 · Radiation Sensitivity Assay (Optional) Day 30-90
  1. a Irradiate Organoids
  2. b Assess Apoptosis Post-Irradiation

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Attribution

This SOP was written by Organthis based on the published method in Hubert et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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