Skip to content
← Back to browse
BRAIN Publication-derived

Human cerebellar organoids as an in vitro 3D model of Group 3 Medulloblastoma

Source Anderle · CIBIO-University of Trento · 10.1158/0008-5472.can-09-2331

👤 Marica Anderle ⏱ 120 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the development and genetic modification of human cerebellar organoids derived from induced pluripotent stem cells (iPSCs) to model Group 3 Medulloblastoma. Wild-type organoids are electroporated with PiggyBac transposase and donor plasmids expressing oncogenes (Gfi1/c-Myc or Otx2/c-Myc) to generate medulloblastoma-like organoids that recapitulate molecular and epigenetic features of human tumors. These modified organoids can be used for disease modeling, therapeutic target validation, and drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

41 steps across 8 phases

iPSC Maintenance and Expansion Day 0 - Day 20
  1. 1 iPSC Culture Initialization
  2. 2 iPSC Dissociation
Cerebellar Organoid Differentiation Day 1 - Day 35
  1. 1 Organoid Formation Initiation
  2. 2 Transfer to Spinning Flask Culture
  3. 3 Organoid Maturation to Day 35
Organoid Electroporation with Oncogenes Day 35
  1. 1 Plasmid Preparation
  2. 2 Prepare Electroporation Mix - Control (Venus Only)
  3. 3 Prepare Electroporation Mix - cMyc/Gfi1 Combination (GM)
  4. 4 Prepare Electroporation Mix - cMyc/Otx2 Combination (OM)
  5. 5 Prepare Electroporation Mix - SMARCA4 Variants
  6. 6 Transfer Organoids to Electroporation Cuvettes
  7. 7 Perform Electroporation
  8. 8 Post-Electroporation Recovery
Organoid Expansion and Characterization (Days 36–57) Day 36 - Day 57
  1. 1 Continue Spinning Flask Culture
  2. 2 Optional: DZNep Drug Treatment (Days 37–57)
  3. 3 Optional: Tazemetostat Drug Treatment (Days 36–41)
  4. 4 Sample Collection for RNA-seq
  5. 5 RNA-seq Library Preparation
  6. 6 RNA-seq Sequencing
Organoid Analysis and Quantification Day 42 - Day 57
  1. 1 Immunofluorescence Sample Preparation
  2. 2 Cryosectioning
  3. 3 Immunofluorescence Staining
  4. 4 Quantitative Cell Analysis
  5. 5 Statistical Analysis
In Vivo Tumor Development and Characterization Day 35 (electroporation) to Day 120+
  1. 1 Organoid Preparation for Injection
  2. 2 Stereotactic Injection into Nude Mice
  3. 3 Post-Operative Care and Monitoring
  4. 4 Sacrifice and Brain Harvest
  5. 5 Brain Cryoprotection and Sectioning
  6. 6 Immunohistochemistry on Tumor Sections
  7. 7 Tumor Tissue Dissociation for DNA Extraction
Molecular Analysis: DNA Extraction and Methylation Profiling Days 60–120
  1. 1 Genomic DNA Extraction from Organoids/Tumors
  2. 2 DNA Methylation Array Preparation
  3. 3 DNA Methylation Array Scanning
  4. 4 Methylation Profile Classification
RNA-seq Data Analysis and Functional Interpretation Days 90–120
  1. 1 RNA-seq Read Quality Control
  2. 2 Read Mapping to Human Genome
  3. 3 Gene Expression Quantification
  4. 4 Differential Gene Expression Analysis
  5. 5 Functional Annotation and Pathway Analysis
  6. 6 Comparison to Published Medulloblastoma Transcriptomes

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

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

This wording is awaiting legal review.

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