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BRAIN Publication-derived

Medulloblastoma Hindbrain Organoids

Full SOP with Academic or a plan

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

Authors: Thomas Willott, James G Nicholson, Yunchen Xiao, Olumide Ogunbiyi, Benjamin Draper, Talisa Mistry, Laura Donovan, Nicolae Radu Zabet, Ashirwad Merve, Sara Badodi, Silvia Marino Duration: 53 days 9 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Willott et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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