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BRAIN Publication-derived

Patient-Derived Glioblastoma Organoid (GBMO) Generation and Characterization

Source Watanabe et al. · The Ohio State University Wexner Medical Center · 10.1101/377606v1.full

👤 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 ⏱ 28 days 📋 12 phases 🧫 Patient-Derived GSC (Glioblastoma)

Abstract

This protocol describes the generation of three-dimensional glioblastoma organoids (GBMOs) from patient-derived glioblastoma stem cells (GSCs) embedded in Matrigel and cultured in a spinning bioreactor with defined differentiation medium. GBMOs self-organize into tissue-like structures that recapitulate the genomic, transcriptomic, metabolic, and architectural features of primary glioblastomas, enabling the study of glioma-intrinsic molecular programs and progenitor populations in the absence of infiltrating immune cells.

Cell source
Patient-Derived GSC (Glioblastoma)
Application
Disease modeling

Protocol overview

51 steps across 12 phases

GSC Isolation and Culture Preparation Ongoing
  1. 1 Cell source procurement and authentication
  2. 2 GSC growth medium preparation
  3. 3 Culture GSCs in low-attachment flasks
  4. 4 NSC culture (control/comparison)
Neurosphere Formation Days 0–7
  1. 5 Dissociate and aggregate GSCs or NSCs
Matrigel Embedding and Initial Gelation Days 4–8
  1. 6 Prepare cold Matrigel droplets
  2. 7 Allow Matrigel to gel at 37°C
  3. 8 Remove organoid droplets from Parafilm
Bioreactor Culture and Differentiation Days 8 onwards
  1. 9 Stationary culture phase (Days 4–8 in bioreactor)
  2. 10 Prepare organoid differentiation medium
  3. 11 Transfer organoids to spinning bioreactor with differentiation medium
  4. 12 Long-term bioreactor culture
Organoid Harvest and Tissue Processing End of culture (typically Days 21–28 or later)
  1. 13 Harvest organoids from bioreactor
  2. 14 Fixation for histology and immunofluorescence
  3. 15 Wash fixed tissues
  4. 16 Cryoprotection in sucrose
  5. 17 Embed in OCT and cryosection
Histology and Immunofluorescence Staining After tissue sectioning
  1. 18 Hematoxylin and eosin (H&E) staining
  2. 19 Permeabilization and blocking for immunofluorescence
  3. 20 Primary antibody incubation
  4. 21 Secondary antibody incubation
  5. 22 Nuclear counterstaining and mounting
  6. 23 Confocal microscopy imaging
Quantitative Immunostaining Analysis After imaging
  1. 24 Quantify proliferation and apoptosis markers
  2. 25 Measure organoid layer thickness
  3. 26 Regional quantification of marker expression
RNA Extraction and Real-Time Quantitative PCR After organoid harvest or during culture
  1. 27 Harvest organoids for RNA extraction
  2. 28 RNA extraction
  3. 29 Reverse transcription
  4. 30 Quantitative real-time PCR setup
  5. 31 qPCR data analysis
Transcriptome Analysis via Microarray After RNA extraction
  1. 32 Prepare samples for microarray
  2. 33 Hybridize to microarray
  3. 34 Washing and staining
  4. 35 Scan microarrays
  5. 36 Data normalization and filtering
  6. 37 Differential expression analysis
Metabolic Analysis via Seahorse After organoid harvest or during culture
  1. 38 Dissociate organoids for metabolic analysis
  2. 39 Plate cells on Seahorse microplate
  3. 40 Run Seahorse XF Real-Time ATP Rate Assay
  4. 41 Calculate ATP production rates
  5. 42 Statistical analysis of metabolic data
Genomic Analysis – Targeted Sequencing and CNV After organoid harvest
  1. 43 DNA extraction
  2. 44 Library preparation for sequencing
  3. 45 Raw read alignment and processing
  4. 46 Variant calling and annotation
  5. 47 Variant filtering
  6. 48 Copy number variation (CNV) analysis
  7. 49 CNV visualization and interpretation
Optional: Chemotherapeutic Treatment During bioreactor culture (Days 8 onwards)
  1. 50 Temozolomide (TMZ) treatment
  2. 51 Irradiation treatment

Full SOP

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Attribution

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

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