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BRAIN Publication-derived

Glioblastoma Brain Organoids (2024)

Full SOP with Academic or a plan

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

Authors: 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 Duration: 40 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Watanabe et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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