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BRAIN Publication-derived

Glioblastoma Brain Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 28 days 8 phases Cell source: 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

Phase 1 · Patient-Derived GSC Isolation and Culture Day 0 - Day 3
  1. a Tumor Tissue Dissociation
  2. b GSC Culture Initiation
Phase 2 · Cerebral Organoid Generation Day 0 - Day 28
  1. a Embryoid Body Formation
  2. b Neural Rosette Formation – First Round
  3. c Neural Rosette Purification
  4. d 3D Organoid Formation in Matrigel Droplets
  5. e Bioreactor Culture
Phase 3 · GSC Transduction with Fluorescent/Bioluminescent Markers Day 0 - Day 7
  1. a GFP Expression Establishment (Optional)
  2. b Firefly Luciferase Expression (Optional)
  3. c Secreted Luciferase Expression (Optional)
Phase 4 · GSC and Cerebral Organoid Co-Culture (GLICO Formation) Day 0 - Day 14
  1. a Organoid Preparation
  2. b GSC Seeding onto Organoids
  3. c Static Incubation
  4. d Transfer to Organoid Differentiation Medium
  5. e Bioreactor Culture of GLICOs
Phase 5 · Tumor Assessment and Characterization Day 7 - Day 14
  1. a Immunofluorescence Microscopy
  2. b EdU Incorporation Assay (Optional)
  3. c Bioluminescence Imaging
  4. d Two-Photon Microscopy
  5. e Histopathological Evaluation
  6. f Transmission Electron Microscopy (Optional)
Phase 6 · Drug and Radiation Treatment (Optional) Day 5 - Day 14
  1. a Chemotherapy Treatment Setup
  2. b Chemotherapy Response Assessment
  3. c Ionizing Radiation Treatment
  4. d Radiation Response Assessment
Phase 7 · Genetic and Molecular Characterization Day 7 - Day 14
  1. a EGFR Amplification Assessment by FISH
  2. b Phospho-RTK Array Analysis
  3. c Quantitative RT-PCR (qPCR) for GFP Expression
Phase 8 · Calcium Imaging of Tumor Microtubes (Optional) Day 10 - Day 14
  1. a Calcium Indicator Loading
  2. b Time-Lapse Imaging

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Attribution

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

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