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BRAIN Publication-derived

Glioblastoma Brain Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Fan et al., 2024 · Fudan University, Shanghai, China · 10.1016/j.isci.2024.109111

Authors: Qi Fan, Hanze Wang, Tianyi Gu, Huihui Liu, Peng Deng, Bo Li, Hui Yang, Ying Mao, Zhicheng Shao Duration: 105 days 10 phases Cell source: Human ESC (H9) and Patient-Derived GSC (Glioblastoma Stem Cell 3264)

Abstract

This protocol establishes a 3D co-culture model using 3D printed molds and Matrigel to investigate interactions between glioblastoma organoids and region-specific brain organoids (dorsal forebrain, composed of excitatory neurons; ventral forebrain, composed of inhibitory neurons). The model enables visualization of glioblastoma cell invasion, axonal growth modulation, and neuronal activity changes induced by tumor cells.

Cell source
Human ESC (H9) and Patient-Derived GSC (Glioblastoma Stem Cell 3264)
Application
Disease modeling; Glioblastoma-neuron interaction study

Protocol overview

45 steps across 10 phases

Phase 1 · Preparation of 3D Printed Mold Before culture initiation
  1. a Design and 3D Print Mold
  2. b Bond Mold to Petri Dish
  3. c Sterilize and Prepare Mold
Phase 2 · Generation of Dorsal Forebrain Organoids (DO) Days 0-105+
  1. a Dissociate H9 ESCs and Initiate Organoid Formation
  2. b Transfer Organoids to Six-Well Plates
  3. c Switch to Neurobasal Medium (Day 7)
  4. d Modify Medium (Day 21)
  5. e Long-Term Maintenance
Phase 3 · Generation of Ventral Forebrain Organoids (VO) Days 0-105+
  1. a Initiate VO with Ventralization Factors (Days 0-7)
  2. b Continue Ventralization (Day 7-14)
  3. c Transition to Neurobasal Medium (Day 21)
  4. d Switch to Maturation Medium (Day 28+)
Phase 4 · Generation and Maintenance of Glioblastoma Organoids (GB) Ongoing maintenance
  1. a Initiate GSC 3264 Culture
  2. b Maintain Spheroid Culture in Six-Well Plates
  3. c Generate GB Organoids in Matrigel
Phase 5 · 3D Co-culture of Brain Organoids with Glioblastoma Organoids 12-16 days (typically 14 days for RNA-seq)
  1. a Prepare Sterilized Molds
  2. b Position Organoids in Mold
  3. c Initiate Co-culture
  4. d Culture Duration
Phase 6 · Lentiviral Transduction for GFP Labeling Optional; timing depends on experimental design
  1. a Produce Lentiviral Particles
  2. b Transduce Glioma Stem Cells
Phase 7 · Immunofluorescence Staining After organoid generation or co-culture
  1. a Fix Organoids
  2. b Wash and Dehydrate
  3. c Prepare Cryosections
  4. d Block and Incubate with Primary Antibodies
  5. e Incubate with Secondary Antibodies
  6. f Mount and Image
Phase 8 · RNA Sequencing Analysis After 14 days of co-culture
  1. a Extract Total RNA
  2. b Assess RNA Quality and Quantity
  3. c Prepare Libraries and Sequence
  4. d Process Raw Data
  5. e Map Reads to Reference Genome
  6. f Identify Differentially Expressed Genes (DEGs)
  7. g Functional Analysis
Phase 9 · Western Blotting After 14 days of co-culture
  1. a Prepare Protein Samples
  2. b Centrifuge and Quantify Protein
  3. c Prepare and Load Protein Samples
  4. d Electrophoresis and Protein Transfer
  5. e Block Membrane and Incubate with Primary Antibody
  6. f Incubate with Secondary Antibody and Detect
  7. g Quantify Band Intensity
Phase 10 · Microelectrode Array (MEA) Recording After 7 days of co-culture (for 14-week-old organoids)
  1. a Prepare MEA Plates
  2. b Place Organoids on Electrodes
  3. c Record Neural Activity
  4. d Analyze Recordings

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Attribution

This SOP was written by Organthis based on the published method in Fan et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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