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BRAIN Publication-derived

Assembly of glioblastoma tumoroids and cerebral organoids: a 3D in vitro model for tumor cell invasion

Source Kim et al., 2025 · Seoul National University College of Medicine · 10.1002/1878-0261.13740

👤 Jieun Kim, Rokhyun Kim, Wonseok Lee, Gyu Hyun Kim, Seeun Jeon, Yun Jin Lee, Jong Seok Lee, Kyung Hyun Kim, Jae-Kyung Won, Woochan Lee, Kyunghyuk Park, Hyun Je Kim, Sun-Wha Im, Kea Joo Lee, Chul-Kee Park, Jong-Il Kim, Ji Yeoun Lee ⏱ 40 days 📋 10 phases 🧫 Patient-Derived GBM Tumoroids and Human iPSC-Derived Cerebral Organoids

Abstract

This protocol establishes a glioblastoma-cerebral organoid assembloid (GCOA) model by fusing patient-derived GBM tumoroids with hiPSC-derived cerebral organoids to generate a 3D in vitro system that recapitulates glioblastoma invasion into normal brain tissue. The model enables visualization of single- and collective-cell invasion patterns, tumor microtube formation, and tumor–normal-cell interactions in a tissue-like microenvironment.

Cell source
Patient-Derived GBM Tumoroids and Human iPSC-Derived Cerebral Organoids
Application
Disease modeling (glioblastoma invasion and tumor-microenvironment interaction study)

Protocol overview

55 steps across 10 phases

GBM Tumoroid Culture Establishment 0–30+ days
  1. 1 Tissue dissociation
  2. 2 Cell suspension preparation
  3. 3 Matrigel embedding and plating
  4. 4 Initial tumoroid culture
  5. 5 Passage tumoroids
Lentiviral GFP Labeling of GBM Cells 0–3 days prior to assembly
  1. 1 Lentiviral vector production
  2. 2 GBM cell infection
  3. 3 Maintenance of labeled tumoroids
Cerebral Organoid (CO) Generation 0–40 DIV (days in vitro)
  1. 1 hiPSC culture preparation
  2. 2 Embryoid body (EB) formation
  3. 3 Neural induction
  4. 4 Maturation phase
GCOA Assembly and Co-culture 0–30+ days after fusion (DAF)
  1. 1 Preparation of organoids for fusion
  2. 2 Co-culture initiation
  3. 3 Transfer to culture plates
  4. 4 Ongoing GCOA maintenance
Live Imaging of Tumor Cell Invasion 3–20 DAF recommended
  1. 1 Sample preparation for live imaging
  2. 2 Time-lapse image acquisition
  3. 3 Data analysis
Immunohistochemistry and Immunofluorescence Staining 14–30 DAF
  1. 1 Sample fixation for histology
  2. 2 Paraffin embedding and sectioning (for IHC)
  3. 3 IHC staining procedure
  4. 4 Immunofluorescence (IF) staining
  5. 5 Secondary antibody and staining completion
  6. 6 Image acquisition
Whole-Mount Staining and Tissue Clearing 14 DAF
  1. 1 GCOA fixation
  2. 2 Blocking and permeabilization
  3. 3 Primary antibody incubation
  4. 4 Washing and secondary antibody incubation
  5. 5 Final wash and fixation
  6. 6 Dehydration
  7. 7 Refractive index matching
  8. 8 3D imaging of cleared GCOAs
Correlative Light and Electron Microscopy (CLEM) 14 DAF
  1. 1 Sample fixation for electron microscopy
  2. 2 Vibratome sectioning
  3. 3 Confocal imaging of regions of interest (ROIs)
  4. 4 Tissue dissection and sample preparation
  5. 5 Serial section collection
  6. 6 SEM imaging
  7. 7 Image alignment and 3D reconstruction
RNA Extraction and qPCR 14 DAF
  1. 1 mRNA extraction
  2. 2 cDNA synthesis
  3. 3 qPCR setup and amplification
  4. 4 Data analysis
RNA Sequencing and Transcriptomic Analysis 14 DAF (sample collection and preparation)
  1. 1 GCOA dissociation and cell sorting
  2. 2 FACS sorting of GFP+ tumor cells
  3. 3 RNA extraction and quality assessment
  4. 4 Library construction
  5. 5 Sequencing
  6. 6 Raw data quality control
  7. 7 Read processing and alignment
  8. 8 Gene expression quantification
  9. 9 Differential expression analysis
  10. 10 Gene ontology and pathway analysis
  11. 11 Cellular state estimation

Full SOP

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Attribution

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

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