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BRAIN Publication-derived

Sonic Hedgehog Medulloblastoma Hindbrain Organoids

Full SOP with Academic or a plan

Source van Essen et al., 2025 · Nuffield Department of Clinical Neurosciences, University of Oxford Medical Sciences Division · 10.1093/noajnl/vdae218

Authors: Max J. van Essen, Alina Nicheperovich, Benjamin Schuster-Böckler, Esther B. E. Becker, John Jacob Duration: 60 days 5 phases Cell source: Human iPSC-derived cerebellar organoids co-cultured with SHH-MB cell lines (DAOY, ONS-76)

Abstract

This protocol describes the differentiation of human iPSC-derived cerebellar organoids and their co-culture with sonic hedgehog medulloblastoma (SHH-MB) cell lines to model the tumor microenvironment. The co-culture system allows investigation of how the nonmalignant microenvironment regulates malignant cell states and transcriptional phenotypes using single-cell RNA sequencing.

Cell source
Human iPSC-derived cerebellar organoids co-cultured with SHH-MB cell lines (DAOY, ONS-76)
Application
Disease modeling of sonic hedgehog medulloblastoma; microenvironment-tumor cell interaction studies

Protocol overview

28 steps across 5 phases

Phase 1 · Cerebellar Organoid Differentiation Day 0-35+
  1. a iPSC Dissociation and Cell Preparation
  2. b Plating of Organoid Initiation
  3. c FGF2 Supplementation
  4. d Medium Change at Day 7
  5. e Full Medium Change and Transfer at Day 14
  6. f Switch to Differentiation Medium at Day 21
  7. g Full Medium Changes at Days 28 and 35
  8. h Maintenance Beyond Day 35
Phase 2 · Medulloblastoma Cell Line Culture and Tumor Spheroid Generation Day 0-7
  1. a DAOY Cell Line Maintenance
  2. b ONS-76 Cell Line Maintenance
  3. c Tumor Spheroid Generation from DAOY and ONS-76
  4. d Spheroid Maturation
Phase 3 · Organoid–Tumor Spheroid Co-culture Setup and Maintenance Day 35-60 (organoid) + 1-25 (co-culture)
  1. a Co-culture Initiation (Day 35 Organoid + Day 7 Spheroid)
  2. b Facilitate Fusion by Angled Incubation
  3. c Co-culture Maintenance (Days 2-25)
  4. d Parallel Controls
Phase 4 · Single-Cell Isolation and Sample Preparation Day 60 (organoid + 25-day co-culture)
  1. a Harvest Co-culture Samples
  2. b Enzymatic Dissociation of Organoids
  3. c Wash and Trituration of Organoids
  4. d Dead Cell Removal
  5. e Cell Viability Assessment and Counting
  6. f Resuspension in Sequencing Buffer
  7. g Enzymatic Dissociation of Tumor Spheroid Monocultures
  8. h Processing of Tumor Spheroid Cells
  9. i Processing of Monolayer Cell Cultures
Phase 5 · Single-Cell RNA Sequencing Sample processing day
  1. a Single-Cell Barcoding and cDNA Synthesis
  2. b Library Preparation and Amplification
  3. c Illumina NovaSeq 6000 Sequencing

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Attribution

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

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