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BRAIN Publication-derived

Medulloblastoma Brain Models

Full SOP with Academic or a plan

Source Contenti et al., 2023 · Université Côte d'Azur, INSERM U1065, C3M, Nice, France · 10.1038/s41419-023-06275-0

Authors: J. Contenti, Y. Guo, A. Mazzu, M. Irondelle, M. Rouleau, C. Lago, G. Leva, L. Tiberi, I. Ben-Sahra, F. Bost, N. M. Mazure Duration: 92 days 11 phases Cell source: Human iPSC (cerebellar organoids); Human medulloblastoma cell lines (HDMB-03, D-458)

Abstract

This protocol establishes a comprehensive wet-lab methodology for investigating mitochondrial NADH shuttle pathways (glycerol-3-phosphate shuttle and malate-aspartate shuttle) as therapeutic targets in Group 3 medulloblastoma cells under hypoxic conditions (1% O₂, 6% O₂, and 21% O₂). The protocol encompasses cell culture, pharmacological inhibition studies, metabolic profiling, and validation in human cerebellar organoid-derived tumor models to identify synergistic drug combinations that induce apoptosis in treatment-resistant Group 3 medulloblastoma.

Cell source
Human iPSC (cerebellar organoids); Human medulloblastoma cell lines (HDMB-03, D-458)
Application
Disease modeling and drug screening for Group 3 medulloblastoma metabolic vulnerabilities

Protocol overview

52 steps across 11 phases

Phase 1 · Cell Culture Preparation and Maintenance Days −7 to 0
  1. a Culture HDMB-03 and D-458 medulloblastoma cells
  2. b Prepare hypoxic and normoxic culture conditions
Phase 2 · Pharmacological Inhibitor Treatment Days 0 to 3
  1. a Treat cells with iGP-1 (mGPDH inhibitor)
  2. b Treat cells with AOAA (MAS inhibitor)
  3. c Treat cells with GNE-140 (LDHA inhibitor)
  4. d Treat cells with Phenformin (mitochondrial complex I inhibitor)
  5. e Treat cells with drug combinations
  6. f Prepare control and vehicle-treated samples
Phase 3 · Cell Viability and Proliferation Assessment Days 1 to 3
  1. a Count cells and measure viability at 24 h and 72 h
  2. b Record total cell count and viability percentage
Phase 4 · Metabolic Profiling and Respirometry Days 1 to 2
  1. a Measure two parameters on the analyzer. Record the oxygen consumption rate, abbreviated OCR. Record the extracellular acidification rate, abbreviated ECAR.
  2. b Perform sequential metabolic inhibitor injection protocol
  3. c Normalize OCR and ECAR to protein content
  4. d Perform Phenotype MicroArray analysis on OmniLog system
  5. e Measure tetrazolium dye reduction kinetically in OmniLog analyzer
  6. f Analyze OmniLog data and perform pathway analysis
Phase 5 · Metabolomic Analysis (Steady-State Metabolite Profiling) Days 1 to 7
  1. a Culture cells for hypoxic metabolite extraction
  2. b Extract metabolites using 80% methanol on dry ice
  3. c Perform two sequential re-extractions of the pellet
  4. d Dry metabolite extracts under nitrogen gas
  5. e Resuspend dried metabolites and prepare for LC-MS/MS
  6. f Quantify 300 endogenous water-soluble metabolites by selected reaction monitoring (SRM)
  7. g Process metabolomics data and perform statistical analysis
Phase 6 · Enzymatic Assays (Optional Supporting Assays) Days 1 to 3
  1. a Measure intracellular glycerol-3-phosphate levels
  2. b Measure NAD/NADH ratio
  3. c Measure intracellular malate levels
Phase 7 · Protein Expression Analysis (Immunoblotting) Days 1 to 3
  1. a Harvest and lyse cells for immunoblotting
  2. b Determine protein concentration by BCA assay
  3. c Separate proteins by SDS-PAGE
  4. d Transfer proteins to PVDF membrane
  5. e Block and incubate with primary antibodies
  6. f Incubate with secondary antibodies
  7. g Visualize bands using ECL and quantify
Phase 8 · Generation and Maintenance of Human Cerebellar Organoids Days −35 to 0 (or Days 1 to 85 from initial differentiation)
  1. a Culture and maintain human iPSCs
  2. b Differentiate iPSCs into cerebellar organoids (Days 0-35)
  3. c Electroporate organoids to introduce oncogenic alleles (Day 35)
  4. d Continue organoid culture post-electroporation (Days 35-71/79)
Phase 9 · Pharmacological Treatment of Tumor Organoids Days 71-85 (from initial differentiation)
  1. a Prepare organoids for drug treatment
  2. b Treat organoids with single-drug and combination therapies
  3. c Monitor organoid response to treatment
Phase 10 · Immunofluorescence and Apoptosis Detection in Organoids Days 14-21 of treatment (days 85-92 of differentiation)
  1. a Fix and permeabilize organoids
  2. b Block non-specific binding
  3. c Incubate with primary antibody against cleaved Caspase-3
  4. d Incubate with secondary antibody
  5. e Stain with Annexin V for apoptosis confirmation
  6. f Mount organoids and perform confocal microscopy
  7. g Generate 3D reconstructions and quantify apoptosis
  8. h Generate merged color images for publication
Phase 11 · Colony-Forming Assay Days 0 to 7
  1. a Plate cells for colony formation
  2. b Fix and stain colonies
  3. c Wash, dry, and count colonies
  4. d Calculate plating efficiency and survival

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Attribution

This SOP was written by Organthis based on the published method in Contenti et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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