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BRAIN Publication-derived

Patient-derived Glioblastoma Organoid (PGO) Development and Characterization

Source Verduin et al., 2023 · Department of Radiation Oncology (Maastro), GROW School for Oncology and Reproduction, Maastricht University Medical Centre · 10.1093/noajnl/vdad152

👤 Maikel Verduin, Linde Hoosemans, Maxime Vanmechelen, Mike van Heumen, Jolanda A.F. Piepers, Galuh Astuti, Linda Ackermans, Olaf E.M.G. Schijns, Kim R. Kampen, Vivianne C.G. Tjan-Heijnen, Buys A. de Barbanson, Alida A. Postma, Danielle B.P. Eekers, Martijn P.G. Broen, Jan Beckervordersandforth, Katerina Staňková, Frederik de Smet, Jeremy Rich, Christopher G. Hubert, Gregory Gimenez, Aniruddha Chatterjee, Ann Hoeben, Marc A. Vooijs ⏱ 90 days 📋 15 phases 🧫 Patient-Derived iPSC (Glioblastoma)

Abstract

This protocol describes the derivation, culture, and characterization of patient-derived glioblastoma organoids (PGOs) from primary human GBM tissue. PGOs are cultured as three-dimensional structures that maintain genetic and phenotypic heterogeneity of the original tumors, and can be used for drug sensitivity testing, whole-exome sequencing, single-cell analysis, and identification of novel treatment resistance mechanisms.

Cell source
Patient-Derived iPSC (Glioblastoma)
Application
Disease modeling, Drug screening, Treatment resistance investigation

Protocol overview

96 steps across 15 phases

Blood and Tumor Tissue Collection and Processing Day 0
  1. 1 Centrifuge blood samples
  2. 2 Aspirate serum and perform RBC lysis
  3. 3 Wash and centrifuge white blood cells
  4. 4 Suspend white blood cells in freezing medium
  5. 5 Macroscopically dissect tumor tissue
  6. 6 Flash freeze and store tumor tissue
Tumor Dissociation and Cell Preparation Day 0–1
  1. 1 Incubate tumor tissue in RBC lysis buffer
  2. 2 Suspend tumor in dissociation medium
  3. 3 Manually shear tumor pieces during incubation
  4. 4 Filter dissociated tumor cells
Organoid Derivation and Embedding in Basement Membrane Extract Day 1–2
  1. 1 Prepare Cultrex RGF basement membrane extract spheres
  2. 2 Allow Cultrex droplets to set
  3. 3 Transfer organoids to culture medium and orbital shaker
PGO Culture Maintenance Day 2–Day 90+
  1. 1 Prepare Neurobasal Medium Complete (NBMc)
  2. 2 Culture organoids in NBMc on orbital shaker
  3. 3 Monitor organoid growth and morphology
PGO Passaging and Dissociation Every 1–4 weeks
  1. 1 Manually shear organoids
  2. 2 Place sheared organoids on ice to dissolve Cultrex
  3. 3 Incubate with TripLE dissociation enzyme
  4. 4 Further mechanical dissociation with glass Pasteur pipette
  5. 5 Resuspend cells in Cultrex and re-embed
Organoid Embedding and Immunofluorescence Analysis As needed (typically after 2–4 weeks of culture)
  1. 1 Treat organoids with pimonidazole for hypoxia detection
  2. 2 Label proliferating cells with EdU
  3. 3 Fix organoids with paraformaldehyde
  4. 4 Cryopreserve organoids in OCT
  5. 5 Prepare frozen sections
  6. 6 Block sections with normal goat serum
  7. 7 Incubate with primary antibodies
  8. 8 Incubate with secondary antibodies and DAPI
  9. 9 Image sections on fluorescence microscope
Multiplex Immunohistochemistry (MILAL) As needed
  1. 1 Fix organoids with formalin
  2. 2 Embed organoids in agarose gel mold
  3. 3 Process for paraffin embedding
  4. 4 Perform Multiple Iterative Labeling by Antibody Neodeposition (MILAL)
  5. 5 Classify single cells based on marker expression
Protein Extraction and Western Blot Analysis As needed
  1. 1 Manually shear organoids
  2. 2 Wash cells with cold PBS
  3. 3 Lyse cells with RIPA buffer
  4. 4 Flash freeze and thaw lysates
  5. 5 Centrifuge lysates
  6. 6 Quantify protein concentration
  7. 7 Perform Western blot analysis
DNA Extraction and Whole-Exome Sequencing (WES) After 2+ months of culture and ≥3 passages
  1. 1 Culture organoids for at least 2 months with ≥3 passages
  2. 2 Extract DNA from organoids and tissues
  3. 3 Measure DNA concentration
  4. 4 Check DNA degradation and contamination
  5. 5 Submit DNA for whole-exome sequencing
  6. 6 Perform bioinformatics analysis
Single-Cell Karyotype Sequencing After 2+ months of culture
  1. 1 Dissociate organoids into single cells
  2. 2 Sort single cells into 384-well plate
  3. 3 Prepare single-cell karyotype sequencing libraries
  4. 4 Sequence single-cell libraries
  5. 5 Perform bioinformatics analysis
TMZ Sensitivity Testing After 3+ days of rest post-dissociation
  1. 1 Dissociate organoids for viability assay
  2. 2 Plate cells in 96-well format
  3. 3 Allow cells to rest for 72 hours
  4. 4 Prepare TMZ dilution series
  5. 5 Treat cells with TMZ for 5 days
  6. 6 Rest cells for 48 hours post-treatment
  7. 7 Measure cell viability with CellTiterGlo 3D
  8. 8 Measure luminescent signal
  9. 9 Normalize data and generate dose-response curves
Chemoradiation Treatment Testing After 3+ days of rest post-dissociation
  1. 1 Dissociate organoids and plate cells
  2. 2 Allow cells to rest for 72 hours
  3. 3 Apply radiotherapy treatment
  4. 4 Apply concurrent TMZ during radiotherapy (if combination treatment)
  5. 5 Maintain cultures post-treatment
  6. 6 Measure cell viability with CellTiterGlo 3D
  7. 7 Analyze treatment response
Targeted Therapy Testing (Osimertinib as Proof-of-Principle) After 3+ days of rest post-dissociation
  1. 1 Dissociate organoids and plate cells
  2. 2 Allow cells to rest for 72 hours
  3. 3 Prepare osimertinib dose series
  4. 4 Treat cells with osimertinib for 72 hours
  5. 5 Measure cell viability with CellTiterGlo 3D
  6. 6 Generate dose-response curves
  7. 7 Perform Western blot analysis for mechanism of action
JNK Inhibitor and TMZ Combination Treatment After 3+ days of rest post-dissociation
  1. 1 Dissociate organoids and plate cells
  2. 2 Allow cells to rest for 72 hours
  3. 3 Initial screening: treat with TMZ, JNK inhibitor (SP600125), or combination
  4. 4 Measure cell viability after 7-day treatment
  5. 5 Dose-escalation: prepare concentration matrices
  6. 6 Treat cells with dose-escalation combinations
  7. 7 Measure cell viability with CellTiterGlo 3D
  8. 8 Normalize data and calculate Bliss scores
TMZ Resistance and RNA Sequencing Analysis Typically 4–6 weeks
  1. 1 Prepare PGOs for sequential TMZ cycles
  2. 2 First TMZ cycle: Treat with 30 μM TMZ for 5 consecutive days
  3. 3 Recover and passage organoids after first TMZ cycle
  4. 4 Second TMZ cycle: Repeat 30 μM TMZ treatment
  5. 5 Recover organoids after second TMZ cycle
  6. 6 Harvest organoids for RNA extraction
  7. 7 Extract RNA using Nucleospin RNA kit
  8. 8 Quantify and assess RNA quality
  9. 9 Submit RNA for bulk RNA sequencing
  10. 10 Bioinformatics: quality control and mapping
  11. 11 Differential gene expression analysis
  12. 12 Gene enrichment analysis using DAVID

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Verduin et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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