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BRAIN Publication-derived

High-Grade Glioma Brain Organoids

Full SOP with Academic or a plan

Source Tripathy et al., 2025 · University of Michigan, Department of Neurosurgery · 10.3390/bioengineering12101121

Authors: Arushi Tripathy, Sunjong Ji, Habib Serhan, Reka Chakravarthy Raghunathan, Safiulla Syed, Visweswaran Ravijumar, Sunita Shankar, Dah-Luen Huang, Yazen Alomary, Yacoub Haydin, Tiffany Adam, Kelsey Wink, Nathan Clarke, Carl Koschmann, Nathan Merrill, Toshiro Hara, Sofia D. Merajver, Wajd N. Al-Holou Duration: 28 days 7 phases Cell source: Patient-Derived High-Grade Glioma (IDH-wildtype, IDH-mutant, H3K27M-mutant)

Abstract

This protocol describes the generation of three-dimensional, multiregional, patient-derived glioma tumor organoids (GTOs) from resected high-grade glioma specimens while preserving transcriptomic and phenotypic heterogeneity. The GTOs are characterized via bulk RNA sequencing, immunofluorescence, and histology, then utilized for high-throughput drug screening to nominate multi-drug regimens and inform personalized treatment strategies.

Cell source
Patient-Derived High-Grade Glioma (IDH-wildtype, IDH-mutant, H3K27M-mutant)
Application
Disease modeling and drug screening for precision oncology

Protocol overview

35 steps across 7 phases

Phase 1 · Tissue Collection and Preparation Day 0
  1. a Intraoperative tumor specimen collection
  2. b Tissue mincing and washing
Phase 2 · Glioma Tumor Organoid Generation and Maintenance Days 0-28
  1. a Plating minced explants in ultra-low attachment culture
  2. b Media replacement
  3. c Monitor organoid formation and assess viability
Phase 3 · Organoid Fixation and Histological Preparation Day 14-28
  1. a Embed organoids in Histogel
  2. b Formalin fixation
  3. c Paraffin embedding
  4. d Deparaffinization and rehydration of tissue sections
Phase 4 · Hematoxylin and Eosin (H&E) Staining Day 28 (following deparaffinization)
  1. a Hematoxylin staining
  2. b Ethanol treatment and eosin staining
  3. c Dehydration and mounting
  4. d Imaging
Phase 5 · Immunofluorescence Staining Day 28 (following deparaffinization)
  1. a Antigen unmasking
  2. b Washing and peroxide quenching
  3. c Blocking
  4. d Primary antibody incubation
  5. e Washing and secondary antibody incubation
  6. f Final washing and mounting
  7. g Confocal microscopy imaging
Phase 6 · Bulk RNA Sequencing and Analysis Day 14-28
  1. a Organoid harvesting and RNA extraction
  2. b RNA quality control
  3. c Library preparation
  4. d Sequencing
  5. e Demultiplexing and data processing
  6. f Transcriptomic deconvolution analysis
Phase 7 · High-Throughput Drug Screening Day 14-28 (organoid generation); Day 0-5 (drug screening)
  1. a Organoid dissociation and cell counting
  2. b Cell seeding in 96-well plates
  3. c Drug preparation and serial dilution
  4. d Drug incubation
  5. e Cell viability assessment
  6. f IC50 calculation and dose-response analysis
  7. g Viability estimation at clinically relevant concentrations
  8. h Drug Sensitivity Score 3 (DSS3) calculation
  9. i Drug panel selection and reporting

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Attribution

This SOP was written by Organthis based on the published method in Tripathy et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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