Skip to content
Back to browse
BRAIN Publication-derived

Glioblastoma Brain Organoids with Gene Editing

Full SOP with Academic or a plan

Source Wang et al., 2024 · German Cancer Research Center (DKFZ), Heidelberg, Germany and ShanghaiTech University, Shanghai, China · 10.1038/s41698-024-00500-5

Authors: Changwen Wang, Meng Sun, Chunxuan Shao, Lisa Schlicker, Yue Zhuo, Yassin Harim, Tianping Peng, Weili Tian, Nadja Stöffler, Martin Schneider, Dominic Helm, Youjun Chu, Beibei Fu, Xiaoliang Jin, Jan-Philipp Mallm, Moritz Mall, Yonghe Wu, Almut Schulze, Hai-Kun Liu 6 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of genetically defined glioblastoma-like organoids (LEGOs) from human induced pluripotent stem cells using CRISPR/Cas9-mediated knockout of tumor suppressors (PTEN, TP53, CDKN2A, CDKN2B, NF1). The organoids recapitulate key features of human GBM and are used for comprehensive molecular characterization via multi-omics analysis and drug response profiling to identify genotype-specific treatment strategies.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

51 steps across 6 phases

Phase 1 · iPSC Genome Editing and Clone Selection Days 0-20
  1. a iPSC Culture and Maintenance
  2. b Plasmid Preparation with gRNA Inserts
  3. c Cell Dissociation for Electroporation
  4. d Electroporation of gRNA Plasmids
  5. e Post-Electroporation Recovery
  6. f Puromycin Selection
  7. g Single Colony Seeding and Expansion
  8. h T7 Endonuclease I Screening
  9. i Clone A-Tailing and TA Cloning
  10. j Western Blot Validation
  11. k Pluripotency Verification
  12. l Clone Expansion and Banking
Phase 2 · Cerebral Organoid Differentiation Days 0-15
  1. a iPSC Dissociation and Aggregate Formation (Day 0)
  2. b Medium Refresh (Day 3)
  3. c Transition to Neuroectoderm Medium (Day 5)
  4. d Embryoid Body Maturation and Matrigel Embedding (Day 9)
  5. e PTN Supplementation (First 5 Days of Matrigel Culture)
  6. f Transfer to Orbital Shaker Culture (Day 13)
  7. g Regular Medium Changes
  8. h Morphological Assessment
Phase 3 · Sample Preparation for Molecular Analysis Days 30 onwards (1-month organoids and later)
  1. a Organoid Fixation and Cryopreservation
  2. b Gelatin/Sucrose Embedding
  3. c Cryosectioning
  4. d Sample Collection for Single-Cell RNA Sequencing
  5. e Sample Collection for DNA Methylation Analysis
  6. f Sample Collection for Metabolome and Lipidome Analysis
  7. g Sample Collection for Proteome and Phospho-proteome Analysis
Phase 4 · Drug Screening and Efficacy Testing Days 30-60 (on cultured organoids)
  1. a Luciferase Labeling of iPSC Clones
  2. b Drug Library Preparation
  3. c Pre-Treatment Bioluminescence Imaging
  4. d Drug Treatment with Kinase Inhibitors
  5. e Drug Treatment with Library Compounds
  6. f TMZ Treatment
  7. g Post-Treatment Bioluminescence Imaging
  8. h Data Analysis and Drug Efficacy Determination
  9. i IC50 Determination for Lead Compounds
  10. j Validation on WT Organoids
Phase 5 · In Vivo Xenograft Studies Days 45 onwards (orthotopic injection on day 45)
  1. a Organoid Dissociation for Cell Injection
  2. b Mouse Preparation and Anesthesia
  3. c Stereotactic Injection into Right Striatum
  4. d Post-Operative Care and Monitoring
  5. e Tumor Monitoring
  6. f Brain Collection and Histological Processing
Phase 6 · Immunofluorescence and Immunohistochemistry Analysis Post-histology processing
  1. a Slide Preparation and Antigen Retrieval
  2. b Blocking and Primary Antibody Incubation (Immunofluorescence)
  3. c Secondary Antibody Incubation (Immunofluorescence)
  4. d Mounting and Imaging (Immunofluorescence)
  5. e Blocking and Primary Antibody Incubation (Immunohistochemistry)
  6. f HRP-Conjugated Secondary Antibody Incubation (Immunohistochemistry)
  7. g DAB Visualization and Counterstaining (Immunohistochemistry)
  8. h Image Acquisition and Quantification

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence