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BRAIN Publication-derived

Glioblastoma Brain Organoids (No. 2)

Full SOP with Academic or a plan

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

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: 28 days 12 phases Cell source: 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

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

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Attribution

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

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