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BRAIN Publication-derived

Medulloblastoma Brain Organoids for Drug Screening

Full SOP with Academic or a plan

Source Frisira et al., 2019 · Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London · 10.1038/s41419-019-2026-y

Authors: Eleni Frisira, Fatima Rashid, Swastina Nath Varma, Sara Badodi, Valentine Ayodele Benjamin-Ombo, David Michod, Maria Victoria Niklison-Chirou Duration: 28 days 10 phases Cell source: Patient-Derived iPSC (Medulloblastoma G3/G4)

Abstract

This protocol demonstrates the use of NPI-0052, a proteasome inhibitor, alone and in combination with γ-radiation to induce apoptosis in patient-derived medulloblastoma cells and three-dimensional tumor organoids. The treatment synergistically activates apoptosis through ROS generation and p53/p73 stabilization, providing a preclinical model for testing adjuvant therapies in aggressive medulloblastoma subtypes.

Cell source
Patient-Derived iPSC (Medulloblastoma G3/G4)
Application
Disease modeling and drug screening for medulloblastoma treatment

Protocol overview

37 steps across 10 phases

Phase 1 · Cell culture preparation and maintenance Day 0 onwards
  1. a Prepare DMEM + GlutaMAX culture medium
  2. b Prepare DMEM:F12 culture medium with growth factors
  3. c Culture cells in humidified CO2 incubator
Phase 2 · NPI-0052 preparation and cell treatment Day 0-1
  1. a Dissolve NPI-0052 in DMSO
  2. b Prepare DMSO vehicle control
  3. c Treat cells with NPI-0052 at indicated concentrations
Phase 3 · γ-radiation treatment Day 1
  1. a Irradiate cells or organoids with γ-radiation
  2. b Return cells to incubator post-irradiation
Phase 4 · Combination treatments and timing schemes Day 1-2
  1. a Pre-treatment protocol: NPI-0052 followed by γ-radiation
  2. b Post-treatment protocol: γ-radiation followed by NPI-0052
Phase 5 · p73 knockdown via siRNA Day 0-3
  1. a Prepare siRNA transfection complex
  2. b Incubate transfected cells for 48 hours
Phase 6 · Tumor organoid generation and culture Day 0-28
  1. a Prepare tumor cell-Matrigel suspension
  2. b Plate Matrigel-cell droplets on parafilm mould
  3. c Allow Matrigel droplets to set
  4. d Transfer organoids to tissue culture dish with medium
  5. e Static culture phase (4 days)
  6. f Transfer organoids to orbital shaker
  7. g Continued organoid culture with medium changes
Phase 7 · Apoptosis detection via Annexin V-FITC Day 1-2
  1. a Harvest treated cells
  2. b Perform Annexin V-FITC staining
  3. c Analyze by flow cytometry
Phase 8 · Cell viability assessment Day 1-2
  1. a Treat cells with NPI-0052 and/or radiation
  2. b Harvest treated cells
  3. c Perform CellTiter-Glo Luminescent Cell Viability Assay
  4. d Record luminescence
Phase 9 · Oxidative stress assessment Day 1
  1. a Measure mitochondrial membrane potential via DiOC6(3) staining
  2. b Measure hydrogen peroxide levels
  3. c Measure total glutathione and GSH/GSSG ratio
  4. d Antioxidant rescue experiment
Phase 10 · Immunostaining and histological analysis of organoids Day 1-3
  1. a Treat tumor organoids
  2. b Harvest organoids
  3. c Fix organoids for paraffin embedding
  4. d Section paraffin-embedded organoids
  5. e Perform immunostaining
  6. f Digitize histological slides
  7. g Quantify immunostaining

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Attribution

This SOP was written by Organthis based on the published method in Frisira et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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