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BRAIN Publication-derived

Glioblastoma Cerebral Assembloids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 40 days 10 phases Cell source: 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

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

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Attribution

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

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