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BRAIN Publication-derived

Precision Oncology for High-Grade Gliomas: A Tumor Organoid Model for Adjuvant Treatment Selection

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

👤 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 ⏱ 28 days 📋 7 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Tripathy et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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