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BRAIN Publication-derived

Modelling medulloblastoma pathogenesis and treatment in human cerebellar organoids

Source Willott et al., 2025 · Brain Tumour Research Centre, Blizard Institute, Queen Mary University of London · 10.1101/2024.08.14.607977;

👤 Thomas Willott, James G Nicholson, Yunchen Xiao, Olumide Ogunbiyi, Benjamin Draper, Talisa Mistry, Laura Donovan, Nicolae Radu Zabet, Ashirwad Merve, Sara Badodi, Silvia Marino ⏱ 53 days 📋 9 phases 🧫 Human EPSC (from dura mater fibroblasts)

Abstract

This protocol describes the generation of human cerebellar organoids (CbO) from expanded potential stem cells (EPSC) to model Group 3/4 medulloblastoma (MB) pathogenesis and treatment. CbOs recapitulate human cerebellar development and contain MB cells-of-origin populations; they can be genetically engineered for tumor onset modeling or co-cultured with MB cells for drug testing.

Cell source
Human EPSC (from dura mater fibroblasts)
Application
Disease modeling; Drug screening

Protocol overview

36 steps across 9 phases

EPSC Culture and Expansion Routine passaging prior to organoid initiation
  1. 1 Culture EPSC lines on Geltrex-coated plates
  2. 2 Passage EPSC using 0.1 mM EDTA with Y-27632 supplement
Cerebellar Organoid Initiation (Days 0–2) Days 0-2
  1. 1 Dissociate EPSC and seed in ultra-low attachment plates
  2. 2 Replace media on Day 1
  3. 3 Switch to growth-factor-free chemically defined media on Day 2
Neuroepithelial Induction (Days 2–7) Days 2-7
  1. 1 Add FGF2 and SB431542 to gfCDM
  2. 2 Maintain organoids in 96-well plates with gentle agitation
Cerebellar Specification (Days 7–21) Days 7-21
  1. 1 Transfer organoids to 6-well ultra-low attachment plates on Day 7
  2. 2 Maintain reduced FGF2 and SB431542 from Days 7–14
  3. 3 Introduce FGF19 from Days 14–21
Neuronal Differentiation and Maturation (Days 21–35) Days 21-35
  1. 1 Switch to complete neurobasal media on Day 21
  2. 2 Add SDF1 from Days 28–35
  3. 3 Maintain orbital shaking and perform 50% media changes every 2–3 days
Extended Maturation and Analysis Timepoints (Days 35–49) Days 35-49
  1. 1 Maintain complete neurobasal media without SDF1
  2. 2 Monitor organoid morphology
  3. 3 Harvest organoids for analysis at Days 35 and 49
Genetic Engineering of EPSC for MB Modeling (Optional) Prior to organoid initiation
  1. 1 Produce lentiviral constructs with Tet-inducible system
  2. 2 Transduce EPSC with promoter-driven rtTA construct
  3. 3 Co-infect with TRE-c-MYC-GFP construct
  4. 4 Confirm retention of EPSC markers in transduced cells
  5. 5 Initiate organoid protocol with engineered EPSC
  6. 6 Induce c-MYC expression with doxycycline at Day 35
  7. 7 Monitor c-MYC-GFP expression and maintain doxycycline for 4 weeks
  8. 8 Include control organoids
  9. 9 Harvest edited organoids at Day 63 for analysis
Co-culture of MB Cells with Cerebellar Organoids (CbO-MB) (Optional) Days 35–49 (14 days of co-culture)
  1. 1 Prepare GFP-tagged MB cell lines
  2. 2 Transfer the Day 35 cerebral organoid to ultra-low attachment U-bottomed plates with 96 wells.
  3. 3 Add MB cells in mixed media
  4. 4 Replace half the media on Day 36
  5. 5 Transfer co-cultures to 24-well plates on Day 37
  6. 6 Remove non-infiltrating cells on Day 40
  7. 7 Maintain co-cultures on orbital shaker for remaining days
  8. 8 Harvest CbO-MB at Day 49 for analysis
Drug Treatment of CbO-MB Co-cultures (Optional) Days 49–53 (4 days of drug treatment)
  1. 1 Initiate drug treatment at Day 49 (14 days post-MB cell seeding)
  2. 2 Perform drug and media top-up on Day 51
  3. 3 Harvest treated CbO-MB at Day 53

Full SOP

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Attribution

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

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