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BRAIN Publication-derived

GBM Stem Cell Modified Organoid Protocol (GBMO)

Source Watanabe et al., 2024 · UConn Health Brain and Spine Institute · 10.1093/pnasnexus/pgae051

👤 Fumihiro Watanabe, Ethan W. Hollingsworth, Jenna M. Bartley, Lauren Wisehart, Rahil Desai, Annalisa M. Hartlaub, Mark E. Hester, Paula Schiapparelli, Alfredo Quiñones-Hinojosa, Jaime Imitola ⏱ 40 days 📋 7 phases 🧫 Patient-Derived GSC (Glioblastoma Stem Cells)

Abstract

A growth factor-free protocol for generating patient-derived glioblastoma organoids (GBMO) from glioblastoma stem cells (GSCs) that recapitulate early stages of GBM formation without immune or vascular infiltration. GBMO model the preangiogenic/preimmunological stage of tumor development and reveal intrinsic immune-like molecular programs driven by SATB2+ and HOPX+ progenitor populations.

Cell source
Patient-Derived GSC (Glioblastoma Stem Cells)
Application
Disease modeling and tumor microenvironment characterization

Protocol overview

28 steps across 7 phases

GSC Culture and Sphere Formation Day 0–7
  1. 1 GSC Isolation and Authentication
  2. 2 Initial Culture Medium Preparation
  3. 3 Small GSC Sphere Formation
Matrigel Embedding and Organoid Initiation Day 7–9
  1. 1 Matrigel Droplet Preparation
  2. 2 Culture Transition Medium
Organoid Growth and Maturation Day 9–38+
  1. 1 Continuous Growth in Matrigel (Growth Factor-Free)
  2. 2 Organoid Maturation Monitoring
Treatment and Functional Assays (Optional) Day 30–38+
  1. 1 Temozolomide (TMZ) Treatment
  2. 2 Gamma Irradiation Treatment
  3. 3 Combined TMZ and Irradiation Treatment
  4. 4 Interferon-Gamma (IFN-γ) Functional Assessment
Histology and Immunofluorescence Day 30–40 (any timepoint)
  1. 1 Organoid Fixation
  2. 2 Cryoprotection and Embedding
  3. 3 Cryosectioning
  4. 4 Hematoxylin and Eosin (H&E) Staining
  5. 5 Immunofluorescence Staining
  6. 6 Confocal Microscopy and Image Analysis
Transcriptomic and Molecular Analysis Day 30–40
  1. 1 RNA Extraction
  2. 2 Reverse Transcription (cDNA Synthesis)
  3. 3 Real-Time Quantitative PCR (qPCR)
  4. 4 Microarray Analysis (GeneChip Human Transcriptome Array 1.0)
  5. 5 Differential Expression Analysis
  6. 6 Targeted Sequencing and Copy Number Variation (CNV) Analysis
  7. 7 Single-Cell RNA-Sequencing (scRNA-seq) Analysis (Optional)
Metabolic Analysis Day 30–40
  1. 1 Organoid Dissociation
  2. 2 Seahorse XF Metabolic Assay Setup
  3. 3 Seahorse XF Real-Time ATP Rate Assay
  4. 4 Metabolic Data Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Watanabe et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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