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BRAIN Publication-derived

Sonic Hedgehog Medulloblastoma Cells in Co-culture with Cerebellar Organoids

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

👤 Max J. van Essen, Alina Nicheperovich, Benjamin Schuster-Böckler, Esther B. E. Becker, John Jacob ⏱ 60 days 📋 5 phases 🧫 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

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

Full SOP

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Attribution

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

This wording is awaiting legal review.

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