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BRAIN Publication-derived

Human cerebral organoids model tumor initiation and infiltration in an autologous astrocyte-supported setting

Source Schickel et al., 2025 · GSI Helmholtzzentrum für Schwerionenforschung GmbH · 10.1016/j.isci.2025.113334

👤 Esther Schickel, Tamara Bender, Leon Kaysan, Simone Hufgard, Margot Mayer, David R. Grosshans, Christiane Thielemann, Insa S. Schroeder ⏱ 150 days 📋 6 phases 🧫 Human ESC (H9/WA09-FI)

Abstract

This protocol describes the generation of cerebral organoids from human embryonic stem cells and their use as a model for brain tumor initiation and infiltration. GFP+/c-MYC-high cells are generated via Sleeping Beauty transposon nucleofection, then either fused with whole organoids as tumor spheres (assembloids) or co-cultured with organoid slices at air-liquid interface. This autologous system models tumor behavior in a physiologically relevant 3D microenvironment with astrocyte support, enabling long-term culture and radiotherapy studies.

Cell source
Human ESC (H9/WA09-FI)
Application
Disease modeling; Tumor initiation and infiltration study

Protocol overview

39 steps across 6 phases

Embryoid Body Formation and Early Organoid Development Days 0–15
  1. 1 Culture H9 ES cells on Laminin-521-coated plates
  2. 2 Dissociate H9 ES cells with ReleSR
  3. 3 Aggregate ES cells into embryoid bodies
  4. 4 Transfer EBs to organoid differentiation medium
  5. 5 Guide neural tube formation with microfilaments and Matrigel embedding
  6. 6 Culture organoids in neural differentiation medium
  7. 7 Replace medium and transition to maturation conditions
Genetic Modification and Tumor Cell Isolation Days 11–50
  1. 1 Prepare nucleofection reagents and plasmids
  2. 2 Nucleofect organoids on day 11 of culture
  3. 3 Culture nucleofected organoids in parallel with normal organoids
  4. 4 Isolate GFP+/c-MYC-high cells by FACS at day 50 or day 80
  5. 5 Expand or cryopreserve isolated GFP+/c-MYC-high cells
Tumor Model Generation—Approach 1: Assembloids (Whole Organoids + Tumor Spheres) Days 50–100
  1. 1 Aggregate GFP+/c-MYC-high cells into tumor spheres
  2. 2 Prepare whole normal sister organoids at day 100
  3. 3 Fuse tumor spheres with whole normal organoids
  4. 4 Culture assembloids with medium refreshment
  5. 5 Monitor assembloid development and tumor cell infiltration
Tumor Model Generation—Approach 2: Organoid Slices + Co-culture with Tumor-like Cells Days 50–150
  1. 1 Prepare organoid slices at day 50
  2. 2 Transfer slices to air-liquid interface (ALI) culture inserts
  3. 3 Culture normal organoid slices for ~7–14 days to stabilize
  4. 4 Add GFP+/c-MYC-high cells directly onto organoid slices
  5. 5 Monitor co-culture dynamics and tumor-like cell behavior
  6. 6 Maintain long-term co-culture for therapy studies
X-ray Irradiation and Therapeutic Response Assessment Variable (upon reaching experimental endpoint)
  1. 1 Prepare tumor models for irradiation
  2. 2 Expose tumor models to calibrated X-ray irradiation
  3. 3 Return irradiated models to normal culture conditions
  4. 4 Sample models at post-irradiation timepoints
  5. 5 Quantify radiation-induced changes in c-MYC and GFAP expression
  6. 6 Assess tumor cell infiltration depth post-irradiation
  7. 7 Measure cell death/necrosis by LDH release
Analytical and Characterization Methods Throughout culture and at harvest
  1. 1 Perform immunofluorescence staining on organoid sections
  2. 2 Extract RNA and perform RT-qPCR
  3. 3 Perform bulk RNA sequencing
  4. 4 Perform single nuclei RNA sequencing
  5. 5 Perform M-FISH karyotype analysis
  6. 6 Perform calcium imaging (Ca2+) on organoid slices
  7. 7 Perform scratch assay for cell migration
  8. 8 Measure LDH release as marker of cell viability and necrosis
  9. 9 Perform optical clearing and light-sheet imaging (optional advanced method)

Full SOP

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Attribution

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

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