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BRAIN Publication-derived

Hypoxic Niche and Stemness

Source Linkous et al. · Howard Hughes Medical Institute / Yale University, USA · 10.1016/j.celrep.2019.02.063

👤 Linkous A, Baber D, Postber B, Bhatt SM, Bhatt AH, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt RS, Bhatt AM, Bhatt JE, Bhatt MN, Fine HA ⏱ 28 days 📋 8 phases 🧫 Patient-Derived Glioma Stem Cells + Normal iPSC Cerebral Organoids

Abstract

Linkous et al. developed the GLICO (Glioma-Cerebral Organoid) co-culture model where patient-derived GBM stem cells are introduced into healthy cerebral organoids, recapitulating diffuse tumor invasion including tumor microtube network formation. This model enables testing of anti-invasion therapeutics in a physiologically relevant 3D brain microenvironment.

Cell source
Patient-Derived Glioma Stem Cells + Normal iPSC Cerebral Organoids
Application
GBM Invasion Modeling

Protocol overview

30 steps across 8 phases

Patient-Derived GSC Isolation and Culture Day 0 - Day 3
  1. 1 Tumor Tissue Dissociation
  2. 2 GSC Culture Initiation
Cerebral Organoid Generation Day 0 - Day 28
  1. 1 Embryoid Body Formation
  2. 2 Neural Rosette Formation – First Round
  3. 3 Neural Rosette Purification
  4. 4 3D Organoid Formation in Matrigel Droplets
  5. 5 Bioreactor Culture
GSC Transduction with Fluorescent/Bioluminescent Markers Day 0 - Day 7
  1. 1 GFP Expression Establishment (Optional)
  2. 2 Firefly Luciferase Expression (Optional)
  3. 3 Secreted Luciferase Expression (Optional)
GSC and Cerebral Organoid Co-Culture (GLICO Formation) Day 0 - Day 14
  1. 1 Organoid Preparation
  2. 2 GSC Seeding onto Organoids
  3. 3 Static Incubation
  4. 4 Transfer to Organoid Differentiation Medium
  5. 5 Bioreactor Culture of GLICOs
Tumor Assessment and Characterization Day 7 - Day 14
  1. 1 Immunofluorescence Microscopy
  2. 2 EdU Incorporation Assay (Optional)
  3. 3 Bioluminescence Imaging
  4. 4 Two-Photon Microscopy
  5. 5 Histopathological Evaluation
  6. 6 Transmission Electron Microscopy (Optional)
Drug and Radiation Treatment (Optional) Day 5 - Day 14
  1. 1 Chemotherapy Treatment Setup
  2. 2 Chemotherapy Response Assessment
  3. 3 Ionizing Radiation Treatment
  4. 4 Radiation Response Assessment
Genetic and Molecular Characterization Day 7 - Day 14
  1. 1 EGFR Amplification Assessment by FISH
  2. 2 Phospho-RTK Array Analysis
  3. 3 Quantitative RT-PCR (qPCR) for GFP Expression
Calcium Imaging of Tumor Microtubes (Optional) Day 10 - Day 14
  1. 1 Calcium Indicator Loading
  2. 2 Time-Lapse Imaging

Full SOP

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Attribution

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

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