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BRAIN Publication-derived

Co-culture of Glioblastoma Organoids with Region-Specific Brain Organoids Using 3D Printed Molds

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

👤 Qi Fan, Hanze Wang, Tianyi Gu, Huihui Liu, Peng Deng, Bo Li, Hui Yang, Ying Mao, Zhicheng Shao ⏱ 105 days 📋 10 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fan et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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